Complex formed by N-linked glycoproteins (SIBLINGS) and Factor H

ABSTRACT

The invention provides methods and compositions for exploiting the discovery that members of the small integrin-binding ligand, n-linked glycoproteins family termed SIBLINGS bind to complement Factor H, and moreover that SIBLINGS proteins, such as BSP, exist in relatively acidic forms. The methods provided can be used to detect SIBLINGS proteins in samples from subjects that are suspected of having tumors or abnormal bone turnover. The invention also provides methods of using SIBLINGS proteins to protect cells from complement mediated lysis. Finally, the discovery allows for the creation of specific binding agents that facilitate the detection of SIBLINGS proteins when they are associated with Factor H.

FIELD OF THE INVENTION

The present invention relates to methods of detecting and utilizingsmall integrin-binding ligand, N-linked glycoproteins (SIBLINGS), aswell as the production and use of recombinant SIBLINGS proteins that canbe used as controls for such detection methods.

BACKGROUND OF THE INVENTION

A. Factor H

Factor H, a 150-kDa protein, is a key regulatory braking mechanism innormal and alternate complement-mediated cell lysis. It dissociates andthereby inactivates the assembled C3 convertase, serves as an essentialaccelerator of Factor I-mediated cleavage of C3b to iC3b, and stericallyinhibits C5 binding to C3b (a prerequisite step for terminal pathwayactivation). The salient structural features of Factor H include 20short consensus repeats (SCRs) that contain four cysteine residuesforming two disulfide bonds per repeat. In addition, each SCR containsat least one conserved tryptophan residue per repeat, and Factor H isknown to interact with several sialic acid-containing proteins.

B. Molecular Characterization of BSP, A Member of the SIBLINGS ProteinFamily

Bone sialoprotein (BSP), also briefly known as BSPII, is aphosphorylated and sulfated glycoprotein that is associated with mostnormal and many pathological mineralized matrices. It is a small(approximate Mr=75,000) integrin-binding protein that supports cellattachment in vitro through both RGD-dependent and RGD-independentmechanisms and has a high affinity for hydroxyapatite. BSP is a memberof the family of small integrin-binding ligand, N-linked glycoproteins(SIBLINGS) family that also includes osteopontin (OPN), dentin matrixprotein (DMP1) and dentin sialophosphoprotein (DSPP). All have similargene structures and are clustered on human chromosome 4.

Bone sialoprotein (BSP) constitutes about 10-15% of the non-collagenousproteins found in the mineralized compartment of young bone (Fisher etel., J. Biol. Chem. 258:12723-727, 1983). Immunolocalization and in situstudies have shown BSP to be produced by osteoblasts, osteocytes, andosteoclasts (the multinucleated cells that resorb bone) (Bianco et al.,Calcif. Tissue Int. 49:421-426, 1991). The areas richest in BSP are thecollagen-poor matrix found between areas of new bone, where bone isdeveloping or turning over. Outside of bone, BSP has been found in othermineralized tissues, such as dentin (Fisher et al., J. Biol. Chem.258:12723-727, 1983), cementum (MacNeil et al., J. Bone Min. Res.9:1597-1606, 1994), and calcifying cartilage of the growth plate (Biancoet al., Calcif. Tissue Int. 49:421-426, 1991). Trophoblasts of thedeveloping placenta also express high levels of BSP (Bianco et al.,Calcified Tissue International, 49:421-426, 1991). While placentaltissue is not usually considered to be a mineralized tissue, late termhuman placentas have hydroxyapatite crystals associated with the agingtrophoblasts.

Using human BSP as a model (Fisher et al., J. Biol. Chem. 265:2347-2351,1990), the protein is first made as a 317 amino acid, 35,000 Da protein.A 16 amino acid leader peptide is removed during synthesis. BSP has nodisulfide bonds and it is nearly uniformly hydrophilic along its length,indicating that the protein is likely to be an extended rod in solution.There are three regions particularly rich in glutamic acids residues(“polyglutamic acid domains”) that have long been thought to govern thehigh affinity of this protein for hydroxyapatite. Recent work withrecombinant fragments, however, shows that BSP's ability to bindstrongly to apatite is found throughout its length (Stubbs et al., BoneMiner. Res. 12:1210-1222, 1997).

Human BSP contains four consensus sequences for N-linkedoligosaccharides, three of which are conserved for all mammalian speciesknown to date. These N-linked and the many O-linked oligosaccharidesmake up approximately 50% of the mass of BSP as it is secreted into thehuman bone matrix (Fisher et al., J. Biol. Chem. 258:12723-727, 1983).Tyrosine sulfation and serine/threonine phosphorylation make up theremainder of the known post translational modifications. There are threetyrosine-rich domains in BSP, the last two of which flank the RGD domainand are subject to sulfation. The presence or absence of the sulfategroups does not appear to change the ability of fibroblasts to attach ina simple in vitro assay (Mintz et al., J. Biol. Chem. 269:4845-4852,1994).

The cDNA BSP sequences for rat (Oldberg et al., J. Biol. Chem.263:19430-19432, 1988), human (Fisher et al., J. Biol. Chem.265:2347-2351, 1990), mouse (Young et al., Mamm. Genome 5:108-111,1994), cow (Chenu et al., J. Bone Miner. Res. 9:417421, 1994), hamster(Sasaguri et al., Direct submission to GenBank, Accession number U65889,1996) and chicken (Yang et al., J. Bone Miner. Res. 10:632-640, 1995)have been published. The human (Kerr, J. M., Fisher, L. W., Termine, J.D., Wang, M. G., McBride, O. W. and Young, M. F. Genomics 17:408-415,1993) and chicken (Yang, R. and Gerstenfeld, L. C., J. Cell. Biochem.64:77-93, 1997) genes have also been published. The human IBSP gene mapsvery close to two other members of this family, within 340 kb of SPPI(osteopontin), and within 150 kb of DMP1 with the order being:cen-DMP1-IBSP-SPPI-tel on chromosome 4 (Aplin et al., Genomics30:347-349, 1995; Crosby et al., Genome 7:149-151, 1996). Mouse Ibsp ison the homologous region of chromosome 5 at 56.0 (Young et al., Mamm.Genome 5:108-111, 1994). Other members of the family are also encoded onchromosome 4, for example dentin phosphoprotein and dentin sialoprotein(DSPP) are cleavage products expressed from a single transcript coded bya gene on human chromosome 4 (MacDougall et al., J. Biol. Chem.272(2):835, 1997).

C. Detection of Human BSP

The detection of human BSP in biological samples has been accomplishedusing polyclonal antibodies directed towards denatured whole BSP,non-denatured whole BSP, or synthetic fragments of BSP. Certain tumorshave been found to ectopically express BSP. For example, BSP has beenfound to be expressed by breast cancer tissue, prostate cancer tissue,lung cancer tissue and thyroid cancer tissue (Bellahcene et al., CancerResearch 54:823-826, 1994; Bellahcene et al., Calcif. Tissue Int.61:183-188, 1998; Bellahcene et al., Calcif. Tissue Int. 61:183-188,1997; and Bellahcene et al., Thyroid 8:637-641, 1998 respectively).Additional studies have shown that BSP mRNA levels are increased inhuman breast cancer tissue as well as cell lines derived from breastcancer tissue (Bellachcene et al., Laboratory Investigation 75:203-210,1996).

The detection of BSP in various tissue samples described above has beenaccomplished through the use of polyclonal antibodies directed to eitherwhole human BSP (LF-6) or synthetic fragments of human BSP, such as thesynthetic fragment comprising amino acids 277-294 (LF-83).

An increased level of BSP in serum has been correlated with the presenceof hyperparathyroidism, Paget's disease, multiple myeloma and breastcancer (Seibel, et al., J. Clinical Endocrinology and Metabolism,81:3289-294, 1996). Elevated levels of serum BSP have also been detectedin subjects suffering from rheumatoid arthritis (Mansson et al., J.Clin. Invest. 95:1071-1077, 1995).

SUMMARY OF THE INVENTION

The present invention stems from the discovery that members of theSIBLINGS family of proteins bind to Factor H and that these proteins canconfer resistance to complement mediated lysis. This discovery alsoallows for the creation of assays that more accurately determine thetotal quantity of SIBLINGS proteins in samples, and for the creation ofmethods of utilizing SIBLINGS proteins to inhibit complement formation.

The present invention also takes advantage of the recognition that thepopulation of BSP (one member of the SIBLINGS family of proteins) inserum contains two distinct sub-populations. The first sub-population ofBSP is relatively non-acidic, and it is the predominant sub-populationfound in normal subjects. The second sub-population of BSP is relativelyacidic and it is found predominantly in subjects with various types oftumors. Accordingly, the invention also provides methods of detectingthe relatively acidic BSP and the relatively non-acidic BSP.Furthermore, these methods can be practiced for example, by usingmonoclonal antibodies specific for either the relatively acidic BSP orby simply separating the different forms of BSP based upon their ioniccharges.

One embodiment of the invention involves detecting at least one memberof the SIBLINGS family of proteins in a manner that Factor H does notinhibit the detection of the SIBLINGS protein. The detection of theSIBLINGS protein can occur in vivo or in vito, for example in thecontext of a sample.

In another embodiment the invention provides methods of detecting theSIBLINGS protein by separating the SIBLINGS protein from theSIBLINGS/Factor H complex and then detecting the SIBLINGS protein.Separation of the SIBLINGS protein from Factor H can be accomplished ina variety of ways, for example by heating the sample in the presence ofreducing agents such as DTT (1,4-dithiothreitol) and β-mercaptoethanol.These methods can also incorporate the use of denaturants such as urea,SDS, and formamide.

Factor H can also be separated from the SIBLINGS protein based upon thedifferences between the ionic charge of the SIBLINGS protein and FactorH. Factor H can also be separated from the SIBLINGS protein by reactinga SIBLINGS/Factor H complex with a specific binding agent.

The specific binding agent can be specific for the portion of theSIBLINGS protein that is exposed in the SIBLINGS/Factor H complex or itcan be specific for the SIBLINGS/Factor H complex itself. When thespecific binding agent is specific for the SIBLINGS/Factor H complexitself it is not necessary to separate the SIBLINGS protein from FactorH prior to detection. Furthermore, in some instances the specificbinding agent can serve to separate and identify the SIBLINGS proteinsimultaneously.

The invention also provides methods of making specific binding agentsthat detect the exposed portion of SIBLINGS proteins when they arecomplexed to Factor H as well as specific binding agents thatspecifically detect the SIBLINGS/Factor H complex. Accordingly, theinvention also provides compositions containing these specific bindingagents.

The methods provided by the invention are particularly useful forscreening for the presence or the recurrence of a tumor, such as aliquid tumor, prostate tumor, thyroid tumor, lung tumor, or breast tumorthat is associated with abnormal SIBLINGS protein production. Themethods can be practiced for example, by testing samples of body fluids,such as blood, serum, or saliva. In some instances the tumor may producea relatively acidic SIBLINGS protein, such as BSP, and therefore, theinvention provides methods of detecting the relatively acidic BSPpopulation.

Accordingly, the invention provides transformed organisms that expressesrecombinant SIBLINGS proteins such as BSP, OPN, DSPP, and DMP1 (rBSP,rOPN, rDSPP, and rDMP1, respectively). This organism can express fulllength SIBLINGS proteins or fragments thereof, more specifically theorganism can express the BSP molecule encoded by the construct shown inSEQ ID NO: 8. This recombinantly produced BSP can then be purified foruse in a variety of different applications.

In another embodiment the invention provides a method of conferringprotection from a complement mediated immune response a subject. Thismethod involves providing a reservoir or other supply in the subjectsbody from which a SIBLINGS protein can be dispersed to interfere withcomplement mediated lysis and inflammation. For example a cell or animplant can be contacted (for example coated) with a SIBLINGS protein.When such a coated implant is introduced into the body, it will provokeless of an inflammatory response. In the case of a cell, a recombinantnucleic acid sequence can be introduced via transformation and the cellwill then express the SIBLINGS protein. This embodiment is useful forprotecting cells that are grafted onto foreign tissue or for protectingbone marrow cells that are being introduced into a foreign host.Alternatively, a reservoir of SIBLINGS protein is placed in the body formetered dispensing of the protein in therapeutic dosages.

In another embodiment of the invention the SIBLINGS protein can bedetected in a subject that is suspected of having abnormal boneturnover, for example abnormal bone turnover associated withosteoporosis.

These and other aspects of the invention will become readily apparent inlight of the description provided below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C are graphs which show the binding of BSP to Factor H asdetected with HP-conjugated avidin after separation on a molecular sievecolumn. FIG. 1A shows a chromatograph of biotinylated BSP. FIG. 1B showsbiotinylated BSP preincubated with normal human serum, and FIG. 1C showsbiotinylated BSP preincubated with Factor H.

FIGS. 2A-C are graphs which show the separation of BSP from Factor Hafter treatment with formamide, DTT, and heat, as detected with LF-100antisera after separation on a molecular sieve column. FIG. 2A shows achromatograph of normal human serum (NHS). FIG. 2B shows biotinylatedBSP-(btBSP) incubated with normal human serum and FIG. 2C shows normalhuman serum after incubation with DTT in the presence of formamide andheat.

FIGS. 3A and 3B are graphs which show the result of a titration ofFactor H with BSP. FIG. 3A shows relative fluorescence of Factor H, andFactor H complexed with BSP. FIG. 3B shows the fractional acceptorsaturation (f_(a)) vs. the molar ratio of BSP to Factor H(C_(s)), thelatter of which was constant throughout the assay. This figure showsthat BSP binds 1 with Factor H and has a binding constant of ≦1 nM.Similar results were seen with OPN and DMP1.

FIGS. 4A-F are graphs which show profiles of serum samples generated byseparating the two sub-populations of BSP on a Toyopearl QAE column.FIGS. 4A and B show two normal sera, FIG. 4C shows the profile from asubject known to have a thyroid tumor. FIG. 4D shows the profile from asubject known to have a lung tumor. FIG. 4E shows the profile from asubject known have a breast tumor, and FIG. 4F shows the profile from asubject known to have a prostate tumor.

FIGS. 5A-E: show profiles from 5 different subjects that were known tohave breast cancer.

FIG. 6 shows the level of serum BSP detected in samples taken from 32individuals under the age of 50, and the serum BSP levels from 36individuals over the age of 50.

FIG. 7 shows the level of serum BSP detected in samples taken fromnormal subjects, subjects known to have prostate cancer, and subjectsknown to have breast cancer.

FIGS. 8A and 8B are graphs which show that recombinantly produced BSPand OPN protect MEL cells from complement mediated lysis. MEL cells wererinsed three times with GVB-MgEGTA buffer, resuspended in GVB-MgEGTA ata density of 5×10⁶ cells/mL, and incubated at 37° C. with differentconcentrations of normal human serum. After 2 hrs, cells were harvestedfor trypan blue exclusion assay. The thiazolium blue assay was carriedout at identical serum dilutions and cell viability was determined byabsorbance at 560 mm. Each data point represents the average of threemeasurements. The error bars represent the standard deviation of themean (A). MEL cells in GVB-MgEGTA buffer were incubated with 10 μg/mL ofeither rBSP or rOPN for 10 minutes at 37° C. Normal human serum was thenadded at a dilution of 1:10 and the cells were returned to 37° C.,incubated for 2 hours and cell viability was determined by trypan blueand thiazolyl blue (MTT) reduction assays (B). Data from 3-7 separateexperiments, with treatment replicates in triplicate, was combined toyield mean values. Error bars represent the standard error of the mean.N=number of experiments combined.

FIG. 9 is a bar graph which shows the involvement of integrin and CD44in protection from complement mediated lysis. MEL cells prepared as inFIG. 8 were treated with normal rBSP (10 μg/mL), rBSP whose RGD sequencehad been mutated to KAE (10 μg/mL), and either GRGDS (SEQ ID NO: 10)peptide (400 μM) followed by rBSP or an αVβ3 antibody (1:4000) followedby rBSP for 10 minutes prior to the addition of normal human serum. Thecells were then incubated for 10 minutes after which cell viabilitydetermined using the MTT assay (A). A cohort of MEL cells werepretreated with rOPN (10 μg/mL) alone, GRGDS (SEQ ID NO: 10) peptidefollowed by rOPN, the αVβ3 antibody followed by rOPN, or an anti-CD44antibody (Chemicon, Co.) followed by rOPN, or hyaluronan (HA) followedby rOPN (B). Cells were then treated with normal human serum andviability assayed as in FIG. 8. Treatment of MEL cells with a pre-formedcomplex of either BSP-Factor H ([BSP+fH]) or OPN-Factor H ([OPN+fH])abolished the protection from 23 complement-mediated lysis. The datarepresents the mean and standard error of the mean for three separateexperiments. Statistical significance was determined by analysis ofvariance. Percent cell viability was determined using A560 absorbancevalues of various conditions and a control where no serum had been added(100% viable). The cross-hatched region represents that range of valuesobserved when normal human serum (1:10) alone was added (maximal celldeath).

FIGS. 10A and 10B are bar graphs which show that human cancer cell linescan be protected from complement mediated lysis through the addition ofSIBLINGS proteins. Human cancer cell lines, MCF-7 (A) and U-266 (B)cells were rinsed three time with GVB-MgEGTA buffer and subsequentlytreated exactly as in the MEL cell complement-mediated cell lysis assaydescribed above except complement active guinea pig serum (GPS) wassubstituted for human serum. Three different dilutions of GPS wereassayed. For cultures pretreated with 10 μg/mL BSP or OPN, the dilutionof GPS used was 1:5. Cell viability was determined by both trypan blueexclusion (solid bar) and MTT (open bar) assay. The bars represent themean values of triplicate samples and the error bars are the standarddeviation values.

FIGS. 11A and 11B are bar graphs which show the separation of DMP1 fromFactor H on a molecular sieve column after treatment with formanide,DTT, and heat, as detected by DMP1 specific polyclonal antisera. FIG.11A shows normal human serum (NHS) and FIG. 11B shows NHS that has beentreated.

SEQUENCE LISTINGS

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1: shows the amino acid sequence of human BSP.

SEQ ID NOS: 2-7: show various amino acid sequences which have been usedas immunogens for the creation of BSP specific antibodies.

SEQ ID NO: 8: shows a cDNA sequence encoding human BSP.

SEQ ID NO: 9: shows an amino acid sequence that was used as an immunogenfor the creation of BSP specific antibodies.

SEQ ID NO: 10: shows the amino acid sequence of a peptide that was usedto compete with proteins from the SIBLINGS family of proteins forintegrin type binding.

SEQ ID NO: 11: shows the nucleic acid sequence of an OPN encodingsequence (Accesion No. J04765).

SEQ ID NO: 12: shows the amino acid sequence derived from the sequenceshown SEQ ID NO: 11.

SEQ ID NO: 13: shows the nucleic acid sequence of an OPN encodingsequence (Accesion No. X13694).

SEQ ID NO: 14: shows the amino acid sequence derived from the sequenceshown SEQ ID NO:13.

SEQ ID NO: 15: shows the nucleic acid sequence of a DMP1 encodingsequence (Accession No. NM_(—)004407).

SEQ ID NO: 16: shows the amino acid sequence derived from the sequenceshown in SEQ ID NO: 15.

SEQ ID NO: 17: shows the nucleic acid sequence of a DSPP encodingsequence (Accession No. AF163151).

SEQ ID NO: 18: shows the amino acid sequence derived from the sequenceshown in SEQ ID NO: 17.

DETAILED DESCRIPTION

I. Abbreviations and Definitions

Abnormal: Deviation from normal characteristics. Normal characteristicscan be found in a control, a standard for a population, etc. Forinstance, where the abnormal condition is a disease condition, such as atumor, a few appropriate sources of normal characteristics might includean individual who is not suffering from the disease (e.g., breastcancer), a population standard of individuals believed not to besuffering from the disease, etc.

Likewise, abnormal may refer to a condition that is associated with adisease. The term “associated with” includes an increased risk ofdeveloping the disease as well as the disease itself. For instance, acertain abnormality (such as an increase in the expression of one ormore SIBLINGS proteins) can be described as being associated with thebiological condition of breast cancer; thus, the abnormality ispredictive both of an increased risk of developing breast cancer and ofthe presence of breast cancer.

Abnormal protein expression, such as abnormal SIBLINGS proteinexpression, refers to expression of a protein that is in some mannerdifferent from expression of the protein in a normal (wildtype)situation. This includes but is not necessarily limited to: (1) amutation in the protein such that one or more of the amino acid residuesis different; (2) a short deletion or addition of one or a few aminoacid residues to the sequence of the protein; (3) a longer deletion oraddition of amino acid residues, such that an entire protein domain orsub-domain is removed or added; (4) expression of an increased amount ofthe protein, compared to a control or standard amount; (5) expression ofan decreased amount of the protein, compared to a control or standardamount; (6) alteration of the subcellular localization or targeting ofthe protein; (7) alteration of the temporally regulated expression ofthe protein (such that the protein is expressed when it normally wouldnot be, or alternatively is not expressed when it normally would be);(8) alteration in post translational processing; and (9) alteration ofthe localized (e.g., organ or tissue specific) expression of the protein(such that the protein is not expressed where it would normally beexpressed or is expressed where it normally would not be expressed),each compared to a control or standard.

Controls or standards appropriate for comparison to a sample, for thedetermination of abnormality, include samples believed to be normal aswell as laboratory values, even though possibly arbitrarily set, keepingin mind that such values may vary from laboratory to laboratory.Laboratory standards and values may be set based on a known ordetermined population value and may be supplied in the format of a graphor table that permits easy comparison of measured, experimentallydetermined values.

Implant: A non-self object that is placed in contact with a subject. Forexample, the object can be an artificial limb, an intraosseous, orintravascular implant or a dental prosthesis. Such objects can comprisemolecules that are generated through biosynthetic pathways, such ashydroxyapatite, or collagen, or the object can contain molecules thatare generated through non-biosynthetic means, such as metal alloys orplastics. Furthermore, such objects can contain a mixture of moleculessuch that some of the molecules are biosynthetically generated and someof the molecules are generated through non-biosynthetic means.

Small Integrin-Binding Ligand, N-Linked Glycoproteins (SIBLINGS): Thefamily of secreted phophoproteins that includes, for example, bonesailoprotein (BSP), osteopontin (OSP), dentin matrix protein (DMP1), anddentin sialophosphoprotein (DSPP). It is also likely that other memberof the SIBLINGS family of proteins will be identified in the future, andthat these additional members of the SIBLINGS family will also bind toFactor H. Hence, when the term SIBLINGS protein is used it refers to notonly to the four family members described above, but also to additionalfamily members that will be identified in the future.

The genes encoding the four members of the SIBLINGS family are locatedon human chromosome 4. Members of the SIBLINGS family of protein areshown, infra, to be capable of binding Factor H. The SIBLINGS/Factor Hcomplex can cause the SIBLINGS protein to be nearly undetectable unlessthe complex is first disrupted. Hence, the discovery that members of theSIBLINGS family of proteins binds to Factor H allows for more accurateand sensitive quantification of the concentration of SIBLINGS proteins.The discovery that SIBLINGS proteins bind Factor H has also led to thedevelopment of methods of using SIBLINGS proteins to selectively protectcells from complement mediated lysis.

Biologically Active SIBLINGS Proteins: Biologically active SIBLINGSproteins are characterized by their ability to protect cells fromcomplement mediated lysis. This activity can be readily assessed usingthe MEL cell assay described below. These proteins can be “derived” fromSIBLINGS proteins such that the endogenous SIBLINGS protein's amino acidsequence is altered to contain amino acid substitutions, deletionsand/or additions. If, however, the endogenous SIBLINGS protein sequenceis altered it will continue to display its ability to inhibitcomplement-mediated lysis and the derived SIBLINGS protein sequence willbe additionally characterized by having at least 50%, 60%, 70%, 80%, or90% sequence identity to the endogenous sequence.

As mentioned above, the inhibition of complement-mediated lysis can bedetected using the MEL cell based assay described below. A SIBLINGSprotein will be found to be “biologically active” if when tested in theMEL assay it confers protection to cells compared to a control samplethat does not contain the SIBLINGS protein.

BSP: The normal endogenous population of bone sialoprotein. Thispopulation contains a wide range of BSP proteins, some that are notextensively post-translationally modified, as well as some of which areextensively post-translationally modified. Furthermore, this normalpopulation contains within it at least two distinct sub-populations. Thefirst sub-population is relatively non-acidic, and it is thesub-population that is most prominent in the serum of normal subjects(subjects without tumors). The second sub-population of BSP isrelatively acidic and found in subjects with tumors, for example insubjects with breast, lung, prostate, and thyroid tumors.

Relatively Acidic BSP: The term relatively acidic BSP refers to asub-population of BSP found primarily in the serum of subjects who havevarious types of tumors. There are multiple methods that can be used todetect the presence of this sub-population in a subject. These methodsinclude column chromatography methods and electrophoresis. Therefore,for clarity the relatively acidic BSP is defined through its behavior onthe specific ion exchange column assay described in section IV, below.Briefly, this section details the use of a Toyopearl QAE column for theseparation of the relatively non-acidic BSP from the relatively acidicBSP. Characterized in this way, and using the assay provided, therelatively acidic BSP will elute from the column at a salt concentrationof greater than 0.5 M NaCl.

While not wishing to be bound to a particular theory, it is believedthat the relatively acidic BSP is post-translationally modified tocontain sialic acids, phosphate, and/or sulfate. In that case, therelative concentration of each sub-population of phosphorylated orsulfated BSP can be determined by assays that quantify the concentrationof sulfur and/or phosphate.

Specific binding agent: An agent that binds substantially only to adefined target. Thus, a SIBLINGS protein specific binding agent bindssubstantially only a particular SIBLINGS protein. For example, the term“BSP specific binding agent” includes anti-BSP and other agents thatbind substantially only to BSP. Similarly, the term “relatively acidicBSP specific binding agent” includes antibodies that are specific forthe relatively acidic BSP and other agents that bind substantially onlyto the relatively acidic BSP. The term “relatively non-acidic BSPspecific binding agent” includes antibodies that are specific for therelatively acidic BSP and other agents that bind substantially only tothe relatively non-acidic BSP. The term “exposed portion of SIBLINGSprotein specific binding agent” includes antibodies and other agentsthat are specific for the exposed portion of a SIBLINGS protein when itis complexed with Factor H.

The discussion below specifically mentions anti-BSP antibodies, howeverit also applies to anti-relatively acidic BSP antibodies,anti-relatively non-acidic BSP antibodies, as well as anti-exposedportion of SIBLINGS protein antibodies.

The term anti-SIBLINGS protein antibodies encompasses monoclonal andpolyclonal antibodies that are specific for SIBLINGS proteins, i.e.which bind substantially only to BSP when assessed using the methodsdescribed below, as well as immunologically effective portions(fragments) thereof. Preferably, the anti-SIBLINGS protein antibodiesused in the present invention are monoclonal antibodies (orimmunologically effective portions thereof) and may also be humanizedmonoclonal antibodies (or immunologically effective portions thereof).Immunologically effective portions of monoclonal antibodies include Fab,Fab′, F(ab′)₂ Fabc and Fv portions (for a review, see Better andHorowitz, Methods Enzymol. 178:476-496, 1989). Anti-BSP antibodies mayalso be produced using standard procedures described in a number oftexts, including Antibodies, A Laboratory Manual by Harlow and Lane,Cold Spring Harbor Laboratory (1988).

The determination that a particular agent binds substantially only tothe desired immunogen may readily be made by using or adapting routineprocedures. One suitable in vitro assay makes use of the Westernblotting procedure (described in many standard texts, includingAntibodies, A Laboratory Manual by Harlow and Lane, Cold Spring HarborLaboratory, 1988). Western blotting may be used to determine that agiven specific binding agent, such as an anti-SIBLINGS proteinmonoclonal antibody, binds substantially only to a SIBLINGS protein, asdescribed in Fisher et al., JBC, 262:9702-08, 1987.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Sample: The term sample includes any artificially generated orbiological specimen that is of interest and is being tested for thepresence of SIBLINGS proteins. More specifically, the term biologicalspecimen includes any specimen that was generated by a living cell.Therefore, samples derived from cells in a tissue culture setting,including the cells themselves fall within the definition or biologicalspecimen. Furthermore, samples taken from a subject, such as tissuesamples and bodily fluids (such a blood plasma or serum) also fallwithin the definition of biological specimen.

Tumor: Tumors are abnormal growths which can be either malignant orbenign, solid or liquid (for example, hematogenous). This termparticularly includes malignant tumors which can be either solid (suchas a breast or prostate carcinoma) or non-solid (such as a leukemia).Tumors can also be further divided into subtypes, such asadenocarcinomas (e.g. of the breast, prostate or lung).

Metastasis: A tumor implant discontinuous with a primary tumor. Anycancer may metastasize to bone, but metastases from carcinomas are themost common. There is a subset of carcinomas that is clinicallyrecognized as more likely to metastasize to bone, and this subsetincludes carcinomas of the breast, lung, prostate, kidney and thyroid.Even if a tumor is not presently recognized as being particularly likelyto metastasize to bone, it can be so categorized by a diagnosis in aparticular subject that a skeletal metastasis has occurred (for exampleby scintigram and biopsy), or by studies of populations of subjectspresenting with a particular histological sub-type of cancer.

Isolated: An isolated biological component (such as a nucleic acid orprotein) has been substantially separated or purified away from otherbiological components in the biological specimen in which the componentnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, and proteins. Nucleic acids and proteins which have been isolatedthus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids and proteins.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified BSPprotein preparation is one in which the BSP is more enriched than theprotein is in its natural environment. That environment being either inserum or within a cell. Preferably, a preparation of BSP is purifiedsuch that BSP represents at least 50% of the total protein content ofthe preparation.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence, if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Subject: This term includes both human and non-human mammals. Similarly,the term subject includes both human and veterinary subjects.

Sequence identity: The term “sequence identity” is used to describe thesimilarity between two nucleic acid sequences or between two amino acidsequences. Sequence identity is typically expressed in terms ofpercentage identity; the higher the percentage, the more similar the twosequences.

Methods for aligning sequences for comparison purposes are well known inthe art. Various programs and alignment algorithms are described in:Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch,J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.USA 85:2444-2448, 1988; Higgins and Sharp, Gene 73:237-244, 1988;Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic AcidsResearch 16:10881-10890, 1988; Huang et al., Computer Applications inthe Biosciences 8:155-165, 1992; and Pearson et al., Methods inMolecular Biology 24:307-331, 1994; Altschul et al., J. Mol. Biol.215:403-410, 1990, presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST™, Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence-analysis programs blastp, blastn, blastx, tblastn and tblastx.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the “Blast 2 sequences” function in the BLAST™ program isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per-residue gap cost of 1). Whenaligning short peptides (fewer than about 30 amino acids), the alignmentshould be performed using the Blast 2 sequences function, employing thePAM30 matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins having even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 45%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% sequence identity.

A first nucleic acid is “substantially similar” to a second nucleic acidif, when optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),nucleotide sequence identity occurs in at least about 60%, 75%, 80%,85%, 90% or 95% of the nucleotide bases. (As used herein, “optimallyaligned” sequences exhibit a maximal possible sequence identity).Sequence similarity can be determined by comparing the nucleotidesequences of two nucleic acids using the BLAST™ sequence analysissoftware (blastn) available from The National Center for BiotechnologyInformation. Such comparisons may be made using the software set todefault settings (expect=10, filter=default, descriptions=500 pairwise,alignments=500, alignment view=standard, gap existence cost=11, perresidue existence=1, per residue gap cost=0.85). Similarly, a firstpolypeptide is substantially similar to a second polypeptide if it showssequence identity of at least about 75%-90% or greater when optimallyaligned and compared using BLAST™ software (blastp) using defaultsettings.

Conservative amino acid substitutions: Conservative amino acidsubstitutions usually have minimal impact on the activity of theresultant protein. Such substitutions are described below.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Such substitutionsgenerally are conservative when it is desired to finely modulate thecharacteristics of the protein. Examples of amino acids which may besubstituted for an original amino acid in a protein and which areregarded as conservative substitutions include: Ser for Ala; Lys forArg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp forGlu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val forLeu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe;Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile orLeu for Val.

More substantial changes in enzymatic function or other features may beobtained by selecting substitutions that are less conservative thanthose above, i.e., selecting residues that differ more significantly intheir effect on maintaining: (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Substitutionsthat generally produce the greatest changes in protein properties arethose in which: (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histadyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions or deletions or additions may be assessedfor SIBLINGS protein derivatives by analyzing the ability of therespective modified polypeptide to inhibit complement-mediated celllysis.

Variant SIBLINGS protein encoding cDNAs or genes may be produced bystandard DNA mutagenesis techniques, for example, M13 primermutagenesis. Details of these techniques are provided in Sam brook etal. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,Ch. 15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al. (ed.) Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience, New York (with periodicupdates), 1987. By the use of such techniques, variants may be createdthat differ slightly from the endogenous SIBLINGS protein encoding cDNAor gene sequences, yet that still encode a biologically active SIBLINGSprotein. DNA molecules and nucleotide sequences that are derivatives ofthose specifically disclosed herein and that differ from those disclosedby the deletion, addition, or substitution of nucleotides while stillencoding a biologically active SIBLINGS protein are comprehended by thisinvention. In their simplest form, such variants may differ from thedisclosed sequences by alteration of the coding region to fit the codonusage bias of the particular organism into which the molecule is to beintroduced.

Additionally, as mentioned above new members of the SIBLINGS family ofproteins can be identified by searching various DNA databases, such asthe database maintained at the National Center for BiotechnoloyInformation in Bethesda, Md. Searches can be done such that sequencesthat are conserved between existing members of the SIBLINGS family ofproteins are used to identify other members of the family. Onceidentified these members can be used to the assays described below todetermine if they maintain SIBLINGS protein biological activity.

Throughout the specification and claims, reference to the singular (suchas “a” or “the”) includes the plural, unless clearly indicated otherwiseby context.

II. Identification and Characterization of SIBLINGS/Factor H Complex

Initial experimentation, which is described below, led to theidentification of a BSP/Factor H complex. The population of BSP in serumfrom subjects displaying tumors was additionally found to contain asub-population of relatively acidic BSP. Subsequent experimentationshowed that both OPN and DMP1 bound to Factor H and it is likely thatthe detection of these SIBLINGS proteins, as well as other members ofthe protein family, will be enhanced by disrupting the SIBLINGS/Factor Hcomplex prior to quantifying protein levels.

A. Identification and Characterization of the BSP/Factor H Complex

BSP is an acidic protein, containing not only many acidic amino acids(particularly glutamic acid) but also phosphate, sulfated tyrosines andoligosaccharides, as well as many sialic acid groups. As such it shouldbind to an anion exchange column under conditions of low salt (<0.1 MNaCl) and neutral pH (6-8). This is indeed the case, and BSP from boneextracts and from culture medium has been found to elute off of a DEAEcolumn at about 0.4 M NaCl in 7 M urea at pH 6.

Surprisingly, serum BSP failed to behave in the same manner as BSP frombone extracts and cell culture medium as described above. In fact,injecting normal human serum onto a DEAE or QAE HPLC column resulted inall of the detectable BSP eluting in the unbound fraction, therefore itbehaved as a neutral or positively charged protein. This indicated thatthe BSP was bound to something that masked its many negative charges,hence attempts were made to disrupt the complex. These attempts involvedusing denaturants, such as 50% formamide or 8 M urea, reducing agentssuch as 1 mM DTT or 10 mM β-mercaptoethanol, and heating to 100° C. for5 minutes. None of these approaches was independently successful indisrupting the complex, as indicated by the failure of the BSP to bindto an anion exchange column.

However, a combination of a denaturant, a reducing agent and heatingfinally did separate the BSP from the complex. This separation was notedby the increased binding of the BSP to an anion exchange column withsubsequent elution in a salt gradient and detection with polyclonalantisera. The anti-sera used was raised in rabbit against human BSP orfragments thereof, and termed LF-6, LF-83, LF-84, LF-100, LF-101,LF-119, LF-120, and LF-125 (see Table 2), which were tested forspecificity via either ELISA assay or Western blotting. (Acta Orthop.Scand, (Suppl. 266) 66:61-65, 1995. The LF-142 (Table 2) polyclonalantisera was raised in rabbit against a synthetic BSP peptide whichcontained several sulfated tyrosines.

The previously uncharacterized protein described above, that was foundto mask the otherwise acidic BSP, was subsequently identified as beingFactor H. The identity of the protein was determined by severaldifferent assay techniques, and these techniques, as well as the resultsare described below.

1) Factor H specific monoclonal antibodies were obtained from Quidel,San Diego, Calif., USA. These antibodies were used to precipitate FactorH from human sera. One sample was left untreated while another washeated to 100° C. for 5 minutes in the presence of 1 mM DTT and 50%formamide. These samples were then separated on a SDS gel and subjectedto western blotting analysis using a BSP specific antibody LF-100.Results from the Western blot analysis showed a high molecular weightband (Mr ^(˜)200,000 Da) in the lane without DTT, and the more typicalBSP size of 75,000 Da in the lane treated with DTT and formamide.

Furthermore, a similar high molecular weight band was also seen when aWestern blot was performed using as a sample purified human Factor H(Quidel) complexed with BSP. This artificially created complex wasimmunoprecipitated by the same Factor H antisera (see above), andanalyzed with the same results as with the whole human serum describedabove. This showed that BSP was bound to Factor H.

2) Biotinylated human BSP was added to normal human serum and thematerial was chromatographed on a calibrated molecular sieve column(Superose 6, Pharmacia, FIG. 1B). A separate chromatogram with thebiotinylated BSP alone was also generated. The biotinylated productswere subsequently detected using HRP-conjugated avidin on a 96-welldirect ELISA (FIG. 1A). The BSP alone chromatographed to the expectedapproximate 75,000 Da position, while the BSP added to the human serumchromatographed to a position consistent with being complexed to FactorH (about 200,000 Da, FIG. 1B, detected with HRP-conjugated avidin).Furthermore, no 75,000 Da, (i.e. free) BSP could be detected, suggestingthat all was bound to the Factor H (Factor H is found at concentrationof about 500 μg/mL human serum). An additional chromatograph wasgenerated with biotinylated BSP added to Factor H (FIG. 1C). Thischromatograph showed that the product eluted at the same size at thesample containing biotinylated BSP and normal human serum (FIG. 1B).

The effect of treatment with DTT and 50% formamide was observed using amolecular sieve column and detecting the products with the polyclonalantibody LF-100. This antibody was previously found to bind to both thebound and unbound form of BSP, however, this binding is probably due tothe fact that the SIBLINGS/Factor H complex is at least partiallydisrupted. Results showed that the BSP from untreated normal human serumeluted at about 200,000 Da (FIG. 2A). Similarly, biotinylated BSP withnormal human serum showed a major peak at about 200,000 Da, the expectedsize of the BSP/Factor H complex, and a second small peak about 75,000Da (FIG. 2B). Finally, a sample of normal human serum incubated in thepresence of 50% formamide and 1 mM DTT at 100° C. for five minutesresulted in a peak corresponding to BSP (FIG. 2C).

3) Biotinylated human BSP bound normally to the anion exchange column.Addition of human serum prior to loading on the anion exchange columncaused the biotin activity to elute in the unbound fraction. Pretreatingwith purified human Factor H gave the same results. These results showedthat Factor H could render the BSP unable to bind to the column, aproperty of the BSP found naturally in human serum.

4) Fluorescence spectroscopic analysis of the tryptophanes of Factor H(BSP does not contain tryptophane) during the binding of BSP shows thatthe two rapidly form a strong interaction that is stoichiometric (1:1)and saturable (FIGS. 3A and 3B).

B. Characterization of OPN and DMP1/Factor H Binding

The salient structural features of Factor H include 20 short consensusrepeats (SCRs) that contain four cysteine residues forming two disulfidebonds per repeat. In addition, each SCR contains one conservedtryptophan residue per repeat and Factor H is known to interact withseveral sialic acid-containing proteins. Therefore, binding interactionsbetween members of the SIBLINGS family of proteins and Factor H can bestudied by steady state fluorescence.

Steady state fluorescence studies were accomplished by titratingpurified human complement Factor H with rBSP, rOPN, and rDMP1 followedby excitation at 295 nm and monitoring emission between 300 and 450 mm.The emission profile of Factor H alone yields a peak at 347 nm. Theaddition of rBSP, rOPN, or rDMP1 in nm increments causes a relativefluorescent intensity quenching. Conversion of the fluorescent intensitytitration into a binding curve by determining the fraction of bindingsites occupied as the fractional change in fluorescence quenching at 347nm yields a saturable binding curve. By steady state fluorescence, thebinding of BSP, OPN, and DMP1, by Factor H are saturable and possess a1:1 stoichiometry and the binding constants are in the nM range. Giventhat normal serum concentration of Factor H is ^(˜)0.5 mg/mL it isprobable that virtually all BSP, OPN, and DMP1 in serum will becomplexed with Factor H.

III. Detection of Elevated Levels of Members of the SIBLINGS Family ofProteins in Serum

Recent observations have shown that BSP and OPN are expressed bymalignant tissue. BSP is expressed in primary breast cancers (Gillespieet al., Int. J. Cancer 73(6):813-815, 1997; Bellahcene et al., CancerRes. 54(11):2823-2826, 1994; Bellahcene et al., Int. J. Cancer69(4):350-353, 1996; Seibel et al., J. Clin. Endocrinol Metab.81(9):3289-3294, 1996), prostate cancer (Waltregny et al., J. Natl.Cancer Inst. 90(13): 1000-1008, 1998), lung cancer (Bellahcene et al.,Calcif. Tissue Int. 61(3):183-188, 1997), thyroid cancer (Bellahcene etal., Thyroid 8(8):637-641, 1998), malignant bone disease (Chen et al.,Histochem. 30(1):1-6, 1998), and by neoplastic odonotoblasts (van derPluijm et al., Cancer Res. 56(8):1948-1955, 1996). In addition, BSPpeptides are potent inhibitors of breast cancer cell adhesion to bone(van der Pluijm et al., Lab Invest. 77(6):665-675, 1997; Tunio et al.,Arch. Pathol. Lab. Med. 122(12):1087-1090, 1998). OPN is expressed inbreast cancer (Gillespie et al., Int. J. Cancer. 73(6):812-815, 1997;Sung et al., Exp. Cell Res. 241(2):273-284, 1998; Bellahcene andCastronovo, Bull. Cancer. 84(1):17-24, 1997; Tuck et al., Int. J. Cancer79(5):502-508, 1998), as well as in prostate cancer (Koeneman et al.,Prostate 39(4)-246-261, 1999), thyroid cancer (Wright et al., Int. J.Cancer. 46(1):39-49, 1990), skin cancer (Senger et al., CBiochim.Biophys. Acta 996(1-2):43-48, 1989) and several other types of cancer(Senger et al., AntiCancer Res. 56(8):1948-1955, 1996; Chambers et al.,Anticancer Res. 12(1):43-47, 1992; Mevoracj et al., J. Exp. Med.188(12):2313-2320, 2331, 1998). Given that these two SIBLINGS proteinsare expressed in cancer tissue it is likely that other members of thefamily will also be found to be over expressed in tumors. Hence, it isdesirable to develop sensitive assays for detecting the presence ofSIBLINGS proteins.

As described above, it has been shown the BSP, OPN, and DMP1 bind toFactor H. Hence, it is likely that these members of the SIBLINGS familyof proteins as, well as the yet untested DPP1 protein, are bound toFactor H in vivo. Therefore, in order to accurately detect theconcentration of these proteins in serum the SIBLINGS/Factor H complexshould be disrupted prior to quantifying the SIBLINGS protein level.

Serum levels of BSP have been quantified previously viaradioimmunoassays (RIAs). These assays, which involve incubation for a24 hour period in the presence of antibody, have led to thedetermination that the circulating levels of BSP in normal subjects arebetween 5 ng/mL and 24 ng/mL (Karmatschek, Clinical Chemistry43:2076-82, 1997). However, using the experimental procedures detailedbelow, which exploit the finding that BSP is at least partiallyundetectable when bound to Factor H, it has been determined that thecirculating concentration of BSP in normal subjects may extend across amuch broader range, for example between 200 ng/mL serum and 1000 ng/mLserum. Thus, the discovery that BSP is bound to Factor H allows BSPlevels to be more accurately determined.

The direct detection of BSP as well as other SIBLINGS proteins in serumis hampered by the fact that it is effectively bound by Factor H and isnot expected to be easily detected by most antisera. For example, serumsamples where eluted from an ion exchange column, and ELISA assays(described below) were preformed using polyclonal antisera LF-6 andLF-83 (Table 2). These antisera were the same polyclonal antisera thatwere used by Bellahcene et al., Cancer Research 54:823-826, 1994;Bellahcene et al., Calcif. Tissue Int. 61:183-188, 1997; Bellahcene etal., Calcif. Tissue Int. 61:183-188, 1998a; and Bellahcene et al.,Thyroid 8:637-641. 1998b, to detect the extopic expression of BSP invarious tumor types. However, these antisera, as well as several othersfailed to detect the BSP/Factor H complex found in serum. Therefore,disrupting the BSP/Factor H complex prior to quantitating serum BSPlevels allows for a more accurate assessment of serum BSP concentration.

There are several different methods which could be used to disrupt theSIBLINGS/Factor H complex and detect serum SIBLINGS protein levels.However, the methods used ideally take into account: 1) the propertiesof the SIBLINGS/Factor H complex compared to the SIBLINGS protein onceit is freed from Factor H; 2) the possible problems resulting fromdenaturing other serum proteins, such as albumin (for example, denaturedserum in 50% formamide is acceptable, but if the denaturant present issubsequently removed, the serum forms a gel even if diluted 1:10 innormal saline buffer); and 3) the SIBLINGS protein should also end up ina medium that is suitable for conducting immunoassays after theSIBLINGS/Factor H complex is disrupted.

One particularly useful method of detecting SIBLINGS proteins involvesseparating the SIBLINGS protein from Factor H, inactivating thecontaminants, such as DTT and BME, and then diluting the sample suchthat the sample is in a media that allows for immunoassay techniques tobe used. This assay method is more feasible, in part, because of theconsiderably higher concentration of SIBLINGS protein that is availableafter separation form Factor H.

Another example of a method for quantifying serum levels of BSP as wellas other SIBLINGS proteins involves: 1) separating the SIBLINGS/Factor Hcomplex from most other serum components, particularly albumin; 2)disrupting the complex (by substantial separation of Factor H from theSIBLINGS protein); and 3) introducing the SIBLINGS protein into bufferconditions compatible with binding to antibodies. The initial separationstep involved diluting 50 μl serum with 150 μl PBS. The resultingdiluted serum was then placed on a disposable anion exchange column(Toyopearl QAE column available from Taso-Haas, a part of Rhom&Hass,Montgomeryville, Pa.). The albumin present in the serum was then boundto the column and the Factor H-SIBLINGS protein complex passed through.The complex was then separated via incubating at 100° C. in the presenceof DTT and formamide. The denaturants and reducing agents were thenremoved by placing the sample on a sephadex G25 column available fromAmersham Pharmacia, Piscataway, N.J., which was previously equilibratedwith normal saline or some other ELISA- or RIA-compatible buffer. Theeluted SIBLINGS protein was then detected by ELISA assay.

Used in this way the invention allows for the development of screeningassays for the detection of SIBLINGS protein in serum. These assaysinvolve separating the SIBLINGS protein from the SIBLINGS protein/FactorH complex and identifying the free SIBLINGS protein. Therefore, theseassays can be used in conjunction with several already existingmonoclonal and polyclonal antisera, as well as other antibodiesdeveloped in the future. This will allow for the more accurateassessment of SIBLINGS protein levels as well as the ability to assessthe presence of several disease states in subjects. For example, thepresence of tumors, Paget's disease, rheumatoid arthritis andhyperparathyroidism can be detected via the above-described assay.

A. Identification of a Highly Acidic Form of BSP in Subjects Known toHave Tumors

As described above, subjects with certain types of tumors have apopulation of BSP which ranges from a peak which elutes at a saltconcentration that is similar to that of normal subjects, to a peakwhich elutes later than BSP from a normal subjects. Therefore, insubjects with tumors (particularly certain types of tumors, such asthose that often metastasize to bone) the serum BSP tends to be not onlyincreased in total amount but also differs from that of a normalsubject. This difference, between the BSP populations of normal subjectsand subjects with tumors, can be exploited for the development of anassay for detecting tumors. One such assay is described below.

Differences in elution profiles between subjects with tumors andsubjects without tumors was illustrated by taking 50 μl of serum fromnormal volunteers and from patients with tumors prior to surgery (nospecial treatment is required for drawing or holding the serum).Subsequently, 50 μl of fresh formamide and 1 μl of 100 mM DTT were addedto the serum sample, and it was heated in a sand-filled heating block(100° C.) for 5 minutes, thereby separating the BSP from Factor H. Thesample was then microfuged for 1 minute at 15,000×g and injected onto a1 mL packed volume Toyopearl QAE (Toso-Haas) column, that had beenpre-equilibrated with 0.05 M sodium phosphate, 50% fresh formamide, pH7.4. The column was then washed using a flow rate of 2 mL/minutes for 5minutes in the same low salt buffer that was used for equilibration, andthen the salt concentration was linearly increased to 2 M NaCl (in thesame buffer, formamide is optional) over 80 minutes (FIG. 4A-F).Finally, the salt concentration was at 2 M NaCl for the final 15minutes.

Similar results were seen in five different subjects with breast tumors.The method used is described above with the exception that a steepersalt gradient was used (25 minutes) followed by 2 M NaCl for the final15 minutes (FIG. 5A-E).

Then 100 μl of each fraction was placed in a corresponding well of amicrotiter plate. It was found that Greiner High-binding plates or NuncMaxisorp worked well for binding both the relatively non-acidic form ofBSP and the relatively acidic form of BSP. The microtiter plates wereincubated for between 1 and 18 hours at 4° C. The plates were thenwashed three times (5 minutes each) with >200 μL/well Tris-bufferedsaline plus 0.05% Tween 20 (TBS-T) and exposed to 100 μL of rabbitpolyclonal antiserum (LF-100) raised against human BSP in TBS-T for 1hour at room temperature. The plates were then washed again as describedabove, and exposed to 100 μL 1:2000 HRP-conjugated goat anti-rabbit IgG(human serum adsorbed, Kirkegaard & Perry, Gaithersburg, Md.) in TBS-Tfor one hour at room temperature. Finally, the plates were washed athird time and the color was developed using 3,3′, 5,5′tetramethylbenzidine and H₂O₂ for 10-30 minutes at room temperature.Color development was stopped by that addition of 25 μL 1 N H₂SO₄ andanalyzed at 450 nm.

Results from the above described HPLC separation technique indicatedthat the relatively acidic BSP was detected only in sera from subjectswith various tumors and was eluted from the column generally at saltconcentrations greater than 1 M NaCl. It was readily apparent thatsubjects with these various tumors not only had increased levels of BSP,but also that they had a different population of BSP compared to that ofnormal subjects.

The use of the semi-quantitative method described above allowed for thedetection of a relatively non-acidic form of BSP that eluted early inthe gradient (≦1 M NaCl). Of course, altering the chromatographyprocedure used, such as by changing the type of resin, could lead toelution of the peak at a different salt concentration, however,regardless of the separation method used there will be a sub-populationof detected BSP which is less negatively charged, and will eluteseparately from the more negatively charged fraction. In normal subjectsthis relatively less negatively charged sub-population would be the onlyor at least the dominant population which is detected. However, in thecase of normal subjects it is still possible that there will be at leasta trace amount of the relatively acidic BSP. It is believed that therelatively non-acidic BSP can be used to measure bone turnovergenerally.

Furthermore, the relatively non-acidic BSP population has been detectedin both normal subjects, as well as in subjects known to have tumors.Subjects who have prostate tumors may have elevated levels of therelatively non-acidic BSP compared to normal, which possibly indicatesthat there is an elevated level of bone turnover.

The above-described assay is designed to detect any tumor that makes BSPand has a sufficiently high tumor load. Using the techniques describedherein, it can be determined whether any particular tumor type isassociated with increased BSP levels, and particularly acidic forms ofBSP. Furthermore, the identification of the relatively acidic form alsoprovides a more sensitive assay than those based on merely assessingtotal serum BSP levels.

Furthermore, the above-described assay could be simplified so that onlythree steps are needed. These steps are, removal of the albumin bybinding de-complexed BSP in low salt (≦0.2 M NaCl), eluting therelatively non-acidic BSP with 1 M NaCl and subsequently eluting therelatively acidic BSP with 2 M NaCl. Samples can then be placed in asolution that allows for antibody binding and the BSP recovered can bedetected by ELISA.

B. Creation of Specific Binding Agents which Recognize Free BSP, as Wellas Bound BSP

Nine rabbit (polyclonal) antisera against human BSP or fragments (LF-6,LF-83, LF-84, LF-100, LF-101, LF-119, LF-120 and LF-125) (includingpeptides) have been made. These include eight polyclonal antibodieswhose activity is summarized in the Acta Orthop. Scand, (Suppl. 266)66:61-65, 1995, as well as one made since (LF-142) that has not beenpublished. The immunogens used to produce these antisera is providedbelow in Table 2. TABLE 2 Gene Anti- Product serum Antigen Known SpeciesHuman BSP LF-6 human bone BSP H, M, D, R, Mou Human BSP LF-83YESENGEPRGDNYRAY H, M, D, R, Mou, C ED- (LPH) (SEQ ID NO: 2) Human BSPLF-84 YESENGEPRGDNYRAY H, M, D, R, Mou, C ED- (LPH) (SEQ ID NO: 2) HumanBSP, LF-100 AIQLPKKAGDIC-(LPH) ′H, M, D, R (SEQ ID NO: 3) Human BSPLF-101 AIQLPKKAGDIC-(CSA) H, M, D, R, P, B (SEQ ID NO: 3) Human BSPLF-II9 recomb. RGDdomain H, M, D, C (AA257-317) (SEQ ID NO: 4) Human BSPLF-120 recomb. Frag I (AA H, M, D, P, R 129--281) (SEQ ID NO: 5) HumanBSP LF-125 recomb. (36-61) H, M, P, R, S (SEQ ID NO: 6) Human BSP LF-142CTVEY*EGEY*EY*TGA H NDY*DNGY*EIY*ESEN Y*(SEQ ID NO: 7)H = human, M = monkey, D = dog R = rat, Mou = mouse, C = chicken, P =pig, S = sheep, B = bovine, LPH = horseshoe crab hemocyanin, CSA =chicken serum albumin*indicates sulfated tyrocineOf these 9 polyclonal antisera only LF-100 can immunoprecipitate naturalBSP, and even LF-100 only binds to a portion of the BSP (<5%) when it iscomplexed with Factor H in human serum as detected by Western blotanalysis. The other antisera precipitate little or no BSP/Factor Hcomplex.

A similar finding was made when an artificial complex was made byincubating an excess of purified Factor H with biotinylated BSP. Onceagain, only the LF-100 antisera was capable of precipitating even asmall portion of the BSP/Factor H complex.

The ability of polyclonal antisera against LF-100 to precipitate bothbound and unbound BSP makes it highly likely that a monoclonal antibodyto the same fragment would behave in the same manner. This monoclonalequivalent to LF-100 can be made by injecting into mice the identical orsimilar peptide-carrier conjugate, as discussed in Example 2. Clonesderived from these mice can also be screened for the ability torecognize both bound and unbound BSP.

It is also believed that a monoclonal antibody against theamino-terminus or carboxy-terminus of BSP will work because thesedomains may remain free of the BSP-Factor H complex.

Alternatively, a monoclonal antibody may be made by first obtaining theBSP/Factor H complex, and then using the complex itself as an immunogen.The BSP/Factor H complex is obtained either through isolating it fromserum or through artificially creating it using isolated Factor H andrecombinant BSP. For example, 5 mg of purified human Factor H (fromQuidel, LaJolla Calif.) can be complexed in vitro with 2 mg of purifiedhuman BSP from the adenoviral system described below. The complex canthen be passed through a QAE column without denaturants to remove anyunbound BSP. The complex can then be chromatographed on a molecularsieve column (Superose 6, Pharmacia) to separate the complex from anyunbound Factor H. The resulting complex is injected into mice formonoclonal production. All clones that can bind to recombinant BSP inthe initial screening can then be assayed to see if they canimmunoprecipitate the BSP/Factor H complex. Even those that can notimmunoprecipitate the intact complex will be useful as BSP monoclonalsfor the detection of BSP after it has been separated from Factor H, orfor use in screening for ectopic expression as described below.

The method of using the BSP/Factor H complex as an immunogen describedabove can also be used with other SIBLINGS protein. Thus, monoclonalantibodies which bind to other SIBLINGS/Factor H complexes can begenerated.

C. Creation of Specific Binding Agents which Recognize the RelativelyAcidic Form of BSP

Monoclonal antibodies that recognize the relatively acidic form of BSPcan be made by using the relatively acidic BSP isolated from humanserum; using relatively acidic BSP isolated from tumor tissue; usingsynthetic peptides which are highly phosphorylated or sulfated; or byusing recombinant BSP. A particularly promising method of producingmonoclonal antibodies to the relatively acidic form of BSP is bycomplexing relatively acidic BSP to Factor H and using the complex as animmunogen. This offers the possibility of recovering an antibody whichrecognizes both the bound and unbound forms of the relatively acidicBSP.

Methods of obtaining the recombinant BSP for use as an immunogen aredescribed below, and methods of obtaining the BSP/Factor H complex foruse as an immunogen are described above.

The relatively acidic form of BSP can be isolated directly from humantumors using the denaturing procedures provided in Fisher et al., J.Biol. Chem. 258:12723-727, 1983. Furthermore, methods of generating suchmonoclonal antibodies are well known to those of skill in the art andthese methods are additionally detailed below. Briefly, subsequent toinjection of the immunogen and fusion, the clones are screened forbinding to the relatively acidic form of BSP, and then subsequentlyscreened for binding to the relatively non-acidic form of BSP.Monoclonal antibodies that specifically detect the relatively acidicform of BSP are selected for subsequent identification of this substancein biological specimens.

D. Detection of Elevated Levels of Serum BSP in Subjects Known to HaveBreast Cancer or Prostate Cancer

Sera were obtained from clinically defined normal, prostate and breastcancer patients (East Coast Biologics, Inc., North Burwick, Me. andJohns Hopkins Bayview Medical Center, General Clinical Research Center)under informed consent with IRB approval and quenched samples were takenfor analysis.

The day before serum was collected a 20 μg/mL solution of recombinantBSP (rBSP) was diluted out from a 1 mg/mL stock solution in 4 Mguanidine HCl. 100 μl of the dilute BSP was added each well of a Greinerhigh binding (product number 655061) 96 well plate. The plate was thencovered and incubated overnight at 4° C., however, shorter incubationperiods are possible.

A solution of TBS-Tween containing 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl,0.05% Tween 20, was prepared. A blocking buffer containing Tris bufferedsaline (TBS)+1% BSA+5% powdered milk was also prepared.

Samples were diluted 1:10 in a 30% formamide 40 mM phosphate buffer, pH7.4. The samples were then reduced with 2 mM dithiothreitol ( 1/100 ofstock, =2.5 μL) at 100° C. for 5 minutes. Residual reducing agent wasinactivated by the addition of equimolar H₂O₂. BSP standards containing2000, 400, 80, 16, 3.2, 0.64, and 0.13 ng/mL rBSP were then diluted out.

LF-100 antibody was then diluted 1:2,000 in TBS-Tween and 100 μl/well ofAb solution was added to polypropylene 96 well 0.5 mL volume plate (USAScientific which is same as Nunc version). 100 μl of standard, or samplewas then added to each well. The lid was then placed on the plate andreacted at room temp, 1 hour.

During incubation of the samples the rBSP coated plate, described above,was removed from the cold and 250 μl blocking buffer was added to eachwell. The blocking buffer was allowed to react for 5 minutes and then itwas removed. The plate was then washed three times with TBS-Tween.

100 μl reaction mixture was added to each well of the rBSP-coated plate.The plate was then incubated for 1 hour at room temp. After incubationthe plate was washed three times using a buffer containing ^(˜)250μL/well TBS-Tween, and then blotted and drained.

The secondary antibody (Goat anti-rabbit IgG (H&L), peroxidase-labeledantibody conjugate, that was human serum adsorbed) which is availablefrom Kirkegaard & Perry, Gaithersburg, Md., was diluted 1:20,000 inTBS-Tween. 100 μL of the secondary antibody was then added to each welland the plates were incubated for 1 hour at room temp. After incubation,the plates were washed three times with ^(˜)250 μL/well TBS-Tween,blotted and then drained.

The results were visualized by providing 100 μL/well TMB (3,3′,5,5″-tetramethylbenzidine) microwell peroxidase substrate (BioFXLaboratories, Randallstown, Md.) and incubating the plates for 30minutes. The reaction was then stopped through the addition of 100 μLstop solution/well and the plates were read at 450 nM.

The results are shown in FIG. 7, and Table 3 below. The meanconcentration of BSP for the 32 subjects with breast cancer was 1170ng/mL, the mean concentration of BSP for the 22 subjects tested withprostate cancer was 1320 ng/mL, and finally the mean concentration ofBSP for the 72 normal subjects was 490 ng/mL. Clearly, most subjectstested in both the prostate group and the breast cancer group haveelevated levels of BSP. Some have BSP levels that are within the normalrange, however, this is expected because not all tumors are thought toexpress BSP.

E. Detection of Elevated Levels of Serum BSP in Subjects Over Age 50

A group of 36 individuals over the age of 50, and a group of 32individuals under the age of 50 were tested to determine their serum BSPlevels. The results are shown in FIG. 6, and Table 3, below. The mean ofthe group under the age of 50 was clearly less than that of the over 50group. This assay, therefore, shows that serum levels of BSP andpossibly other members of the SIBLINGS family of proteins can be used tomeasure bone turnover or other age-related changes. TABLE 3 <50years >50 years All Normal Normals prostate breast Normals BSP mean344.266 619.787 1320 1170 490.13 (ng/mL) Standard 41.318 179.953 289.582364.396 192.096 Deviation Number of 32 36 32 22 68 Individuals Sampled

F. Creation of Cell Cultures Expressing Human BSP in the Highly AcidicForm

Full-length human BSP cDNA (GenBank accession number J05213; Seq. ID NO:8) was excised from the original plasmid with EcoRI and subcloned intothe EcoRI site of the pAC-EF1 adenovirus construct plasmid. Thisconstruct contained the EF-1 promoter and the bovine growth hormone(BGH) 3′ polyadenylation message stabilization sequences, which is amodification of the original pAC vector (Gluzman et al., EucaryoticViral Vectors, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 187-192, 1982). Selection for double cross-over within thereplication-deficient adenovirus genome results in replication-deficientadenovirus that can infect most cell types but can be amplified only inspecific cell culture lines. Infected cells produce the human BSP,modify it according to their specific ability, and secrete the productinto the culture media.

A similar cloning scheme allowed for the production of adenovirusconstructs that encode OPN and DMP1 allowing choice of all lines.

One method of producing the relatively acidic BSP involves human marrowstromal fibroblasts. These cells in primary culture modify the BSP andcan produce the BSP under serum-free conditions for several days. Asmall percentage of the BSP made in these marrow fibroblasts elutes inthe high salt portion of the anion exchange column. Additionally, thisrelatively acidic BSP produced by the fibroblasts infected with theadenoviral vector described above stains a darker blue (characteristicof being more acidic) than other fractions of BSP that are eluted off ofthe anion exchange columns earlier, when using StainsAll dye fordetection((1-ethyl-2-[3-(1-ethylnaptho[1,2d]-thiazolin-2-ylidene)-2-methylpropenyl]naptho[1,2d]thiazolium bromide, made by Eastman Kodak,Rochester, N.Y.).

Additional adenoviral constructs were also made that contain codingregions for OPN and DMP1. These constructs also can be used to expressOPN and DMP1 in various cell types. It is also believed that SIBLINGSproteins, such as OPN and DMP1, also will be found to bepostranslationaly modified such as has been seen in the case of BSP.Hence, the adenoviral vectors that are used to produce OPN and DMP1 canbe used to produce different varieties of postranslationaly modified OPNand DMP1 by simply infecting different cell types. The expressed OPN andDMP1 can then be used in various assays for controls and to generateantibodies.

Transformation of normal fibroblasts with the BSP encoding vector(^(˜)100,000,000 cells on a total of eight 15 cm plates) allowed for thepurification 20-40 mg of human BSP. Amino terminal sequencing verifiedthat the amino-terminus corresponds to that of human BSP. Additionally,LF-83 bound to the product, hence the carboxy-terminus of therecombinant BSP is complete at least through amino acid 295 (of 317).

Furthermore, it is believed that other primary cell lines, as well asother cancer cell lines, transformed with the vector described aboveproduce one or both sub-populations of BSP in cell culture. Severalcancer cell lines are readily available from ATCC and other sources (forexample: MCF-7, CRL-1435, CRL-1470, SAOS-2, MG-63 etc.).

G. Uses of Specific Binding Agents Directed Against the RelativelyAcidic BSP

BSP specific binding agents, as well as other SIBLINGS protein specificbinding agents, such as those made by the methods described herein, canbe used for immunostaining of biopsies and sections of various tumortypes, which can assist in diagnosing and determining the severity ofdisease. BSP levels may also be used to determine possible treatmentsthat would be most suitable for a particular patient. For example, avery strong BSP positive breast cancer biopsy can be used as anindicator that more aggressive treatment is required following surgery(such as adjuvant chemotherapy or radiation therapy). Furthermore,monoclonal antibodies to human BSP may also be useful as immunostainingagents for determining that an otherwise inconclusive biopsy is, infact, cancer.

Examples of BSP specific binding agents are the 13 monoclonal antibodiesthat were produced through the use of a synthetic 27-amino acid peptide(256T-282N). Of the 13 monoclonal antibodies produced 4 are IgG type and9 are IgM type. This peptide was derived from the human BSP sequence,and it was designed such that it contained five tyrosine sulfates andwas conjugated to a carrier protein through an amino terminal Cys(keyhole limpet hemocyanin, KLH). Monoclonal antibodies from miceinjected with this construct were selected if they bound to 1) the samepeptide conjugated to a different carrier protein (bovine serumalbumin); 2) the same peptide, but without the tyrosines sulfationconjugated to the second carrier protein; and 3) recombinant BSP madefrom human marrow fibroblasts infected with the virus constructdescribed above.

H. Use of Specific Binding Agents that Bind to the Portion of BSP thatdoes not Contribute to Increased Activity

This method allows for the detection of the relatively acid BSP. TheBSP/Factor H complex is disrupted and the decomplexed BSP is allowed tobind to an anti-BSP antibody coated plate or bead that specificallybinds to regions of BSP that are not involved in increased acidity.(Examples of such regions are epitopes with the domains used for LF-100or LF-125, but many other likely areas can be easily chosen.) Then asecond binding agent can be added, such as an antiserum, lectin, orother chemical reagent (including those commercially available) whichcan detect various chemical groups that may contribute to the increasedrelative acidity of the BSP in cancer patient serum (phosphoserine,phosphothreonine, sulfated tyrosine, various sialic acids, varioussulfated sugar groups, etc.). This second binding agent binds to the BSPthat was previously bound by the first antiserum. The second antiserumor chemical reagent is then quantified using standard direct or indirectmethods. This approach allows development of a quantitative assay morespecific for the relatively acidic BSP without necessarily relying on asingle antibody system that is specific to the BSP protein. For example,if the increased acidity is determined to be caused by a sialic acidgroup the BSP can be trapped by an antiserum, such as LF-100 or amonoclonal antibody devised to that domain, and then the sialic acid canbe detected and quantified. The post-translational modification itselfmay be common in many proteins in the serum (or urine etc.), however thedetection of the modification on the captured BSP makes the assay usefulfor the detection of a disease.

IV. Inhibition of Complement Cascade Via Expression of Siblings Proteins

A. Results

The key role of Factor H is the regulation of complement and alternatecomplement activity. Factor H can also be ‘hijacked’ by invadingorganisms to permit pathogen evasion of complement mediated lysis.Pathogens such as Streptococcus pyogenes (Kotarsky et al., J. Immunol.160(7):3349-3354, 1998; Jarvis and Vedros, Infect. Immun. 55(1):174-180,1987), Neisseria gonorrhoeae (Ram et al., J. Exp. Med. 188(4):671-680,1999; Ram et al., J. Exp. Med., 187(5):743-752, 1998; Diaz and Sim, J.Immun. 80(3):473482, 1995), and Echinococcus granulosus (Diaz and Sim,J. Immun. 158(8):3779-3786, 1997; Wiles et al., J. Mol. Biol.272(2):253-265, 1997) bind Factor H to their cell surface and areresistant to complement-mediated cell lysis. In addition, molecularmimicry of Factor H occurs when a pathogen makes a protein that issimilar in sequence to Factor H to defend against attack by the hostcomplement system as described in Vaccinia virus (Isaacs et al., Proc.Natl. Acad. Sci. 89(2):628-632, 1992; Huemer et al., Immunology79(4):639-647, 1993), Herpes simplex virus (Donelson et al., MolecularBiol. 91(1):51-66, 1998), and Trypanosoma cruzi (Hall and Joiner, J.Eukaryot Microbiol. 40(2):207-213, 1993; Kipnes and daSilva, Braz. J.Med. Biol. Res. 22(1):1-16, 1989; Ryden et al., Eur. J. Biochem.184(2):331-336, 1989). The following data demonstrates that BSP, DMP1,and OPN play a similar role in tumor cell complement evasion.

The first model system employed was a murine erythroleukemia (MEL) cellline which when incubated with normal human serum can be readily assayedfor alternate complement pathway (ACP)-mediated cell lysis (Varnet etal., Curr. Opin. Cell Biol., 8(5):724-730, 1996). Cell survival wasmeasured by both trypan blue dye exclusion and by thiazolyl blue (MTT)reduction by living mitochondria. Titration with dilutions of normalhuman serum and time courses were carried out to define optimalincubation conditions. At 1:10 dilution, human serum totally lysed theMEL cells as measured by both assay systems (FIG. 8A). The addition ofpurified recombinant BSP to MEL cells followed by treatment with normalhuman serum completely protected the cells from lysis (FIG. 8B).Treatment of MEL cells with recombinant OPN (FIG. 8B) or DMP1 alsoconferred protection from ACP-mediated cell lysis. The protection of MELcells from alternate complement pathway-mediated cell lysis by BSP, OPN,and DMP1 exhibited dose related responses.

The mechanism of protection from complement-mediated lysis wasinvestigated. Preincubation of MEL cells with rBSP in which the RGDsequence had been mutated to KAE completely removed the protectiveaffects of this protein (FIG. 9). Furthermore, preincubation of MELcells with either GRGDS (SEQ ID NO: 10) peptide or an αVβ3 antibody(which blocks that integrin's binding activity) negated the protectiveeffect of rBSP. Up-regulation of the αVβ3 integrin has also been shownto correlate with invasive potential (Culty et al., J. Cell Biol. 111(6Pt 1):2765-2774, 1990). For OPN, pretreatment with GRGDS (SEQ ID NO: 10)peptide or the αVβ3 antibody reduced cell survival, though the magnitudeof reduction was not as great as that seen for BSP (FIG. 9B). OPN hasmultiple potential receptors including αVβ1,β3, β5-containing integrinreceptors as well as CD44, a receptor implicated in attachment, homingand aggregation of lymphocytes as well as neoplastic cells. Pretreatmentof MEL cells with hyaluronan, a natural ligand for CD44 (Faulk andTemple, Nature 262(5571):799-802, 1976), as well as with an anti-CD44antibody also reduced the protective effect of added rOPN (FIG. 9).Treatment of MEL cells with a pre-formed complex of either rBSP-Factor Hor rOPN-Factor H abolished the protection from complement-mediatedlysis. For BSP, this is consistent with immunoprecipitation data thatindicates that the RGD moiety is inaccessible in the solution phaseFactor H complex. Hence, both of these proteins are believed to beentirely masked by Factor H shortly after being secreted by a cell.

Additional assays were conducted to determine if DMP1 could also conferprotection against complement. DMP1 was found to protect the cells fromcomplement-mediated lysis. Preincubation of the cells with antisera toeither αVβ3 integrin or CD44 diminished the DMP1 protection and togetherthey abolished it (p>0.001). Addition of the synthetic GRGDS (SEQ ID NO:10) peptide diminished but did not abolish the protective properties ofDMP1. DMP1 appears to protect the cells from attack by complement bybridging the major complement control protein, Factor H, to cell surfaceintegrins through its RGD motif and through CD44.

To verify that the protective effect of BSP and OPN is exhibited inhuman cancer cells, MCF-7 breast cancer cells selected for non-adherentgrowth and U-266 myeloma cells were used in the alternate complementpathway cell lysis assay with guinea pig serum as the source ofcomplement activity. Both cell types exhibited enhanced survival andprotection from complement-mediated cell lysis when rBSP or rOPN werepresent (FIG. 10). Increasing concentration of guinea pig serum led todecreasing cell viability, while the pretreatment with either 10 μg/mLrBSP or rOPN resulted in increased cell viability.

Collectively the above described results show that DMP1, OPN, and BSPbridge Factor H to integrins and inactivate the destructive complementpathway. Theses results also confirm that OPN and DMP1 bind to CD44.Furthermore, it is likely that other members of the SIBLINGS family willhave the same biological activity.

B. Experimental Procedures

1. Reagents

Rabbit Anti-human BSP antibodies LF-83, LF-100, LF-119, LF-120, LF-125have been previously described (Fisher et al., Bacta Orthop. Scand.Suppl. 266(1):61-65, 1995). Rabbit Anti-BSP peptide-derived antibodyLF-142 and mouse monoclonal antibodies LFmAb-11, LFmAb-2, LFmAb-12, andLFmAb-13 were raised against the sequence EY*EY*TGVNEY*DNGY*EIY*ESENGEP(amino acids 258-285; SEQ ID NO: 9) conjugated to horseshoe crabhemocyanin, where the Y* denotes tyrosine-sulfates. Normal human serum,purified human complement Factor H protein and mouse monoclonal antibodyagainst Factor H were obtained from Quidel Corporation (San Diego,Calif.). Polyclonal antibodies against CD44 and a “functional” antibodyagainst αVβ3 were obtained from Chemicon Co. (Temecula, Calif.).Synthetic purified glycine-arginine-aspartate-serine peptide (GRGDS; SEQID NO: 10) was obtained from Calbiochem-NovaBiochem Corp. (La Jolla,Calif.). Pre-immune serum, human serum adsorbed goat anti-rabbit IgGconjugated to horse radish peroxidase (HRP) as well as goat anti-mouseIgG conjugated to HRP were obtained from Kirkegaard & Perry(Gaithersburg, Md.). HRP-conjugated strepavidin andsulfosuccinimidobiotin were obtained from Pierce Chemical Co. (Chicago,Ill.). Alpha-Minimal Essential Medium (MEM), Dulbecco's ModifiedEssential Medium (DMEM), RPMI 1640, Eagle's Minimal Essential Medium(EMEM), Earle's Balanced salt solution, Hank's balanced salt solutionand heat inactivated fetal bovine serum (FBS) were obtained fromBioFluids, Inc. (Rockville, Md.).

2. Western Blotting

Samples diluted in gel sample buffer were resolved by SDS-PAGE 4-20%gradient gels (Novex Corp, San Diego, Calif.), transferred tonitrocellulose following standard conditions (Towbin et al., Proc. Natl.Acad. Sci. 76(9):4350-4354, 1979). Nitrocellulose membranes were rinsedwith Tris-buffered saline (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl)containing 0.05% Tween 20 (TBS-Tween). After a 1 hour incubation inblocking solution (TBS-Tween+5% non-fat powdered milk) at roomtemperature on rotary shaker, 1:5000 primary antibody was added andincubated overnight at 4° C. The nitrocellulose sheet was washed inTBS-Tween four times for 5 minutes each time with TBS-Tween and then1:50,000 HRP conjugated second antibody in TBS-Tween+5% milk was addedand incubated for 2 hours at room temperature. Following removal of thesecond antibody solution the membrane was washed three times withTBS-Tween and rinsed a final time in enzyme substrate buffer for 5 min.Enhanced chemiluminescence reagents were employed for signal detection(Pierce Chemical Co., Chicago, Ill.) with x-ray film.

3. High Performance Liquid Chromatography

A Shimadzu LC10AS binary gradient system was employed forchromatographic separations. A 1.0 mL packed volume ToyoPearl QAE(TosoHaas, Montgomeryville, Pa.) column was pre-equilibrated with 0.05 Msodium phosphate, pH 7.4, containing 50% fresh formamide. A linear saltgradient increasing to 2.0 M NaCl at 2.0 mL/minute flow rate over 50minutes was employed collecting 1 minute fractions. Size exclusionchromatography utilized a 1.0×30 cm Superose 6 column (AmershamPharmacia, Piscataway, N.J.) equilibrated in 0.05 M sodium phosphate, pH7.4, containing 50% fresh formamide at a flow rate of 0.5 mL/min. Thecolumn was calibrated using commercially available protein standards ofknown molecular weight (Amersham Pharmacia, Piscataway, N.J.).

4. Direct ELISA

Greiner high-binding 96 well plates were coated with 100 μl HPLCfractions overnight at 4° C. Plates were washed three times (5 minuteseach) with TBS-Tween and exposed to 100 μl of 1:2000 primary antibodyfor 1 hour at room temperature. Plates were washed three times andexposed to 100 μl of 1:2000 HRP-conjugated goat anti-rabbit IgG.Following a one hour incubation at room temperature, plates were washedagain three times with TBS-Tween and color was developed using 3,3′,5,5′, tetramethylbenzidine and H₂O₂ for 10 minutes at room temperature.Color development was stopped by the addition of 25 μL 1N H₂SO₄ andanalyzed at 450 nm.

5. Immunoprecipitation

Aliquots of normal human serum diluted 1:100 in immunoprecipitationbuffer (0.1 M Tris, pH 7.2, 0.15 M NaCl, 0.05% Tween 20, and 1%aprotinin) were incubated sequentially with 0.1 mL each of (a) Protein Gagarose (Kirkegaard & Perry; Gaithersburg, Md.), (b) normal rabbit serumIgGs bound to Protein G agarose; (c) rabbit anti-BSP antibodies bound toProtein G agarose. After incubation for 1 hour at 4° C. the reaction wasterminated by centrifugation at 10,000×g for 5 minutes and thesupernatant taken to the next immunoprecipitation. The first twoincubations removed proteins binding non-specifically to agarose and tonormal rabbit serum. The anti-BSP incubation immunoprecipitate wasdissolved in gel sample buffer and analyzed by 4-20% gradient SDS-PAGE.

6. Production of Recombinant SIBLINGS Proteins

Adenoviral constructs encoding BSP (rBSP), DMP1 (rDMP1), and OPN (rOPN)were generated by subcloning BSP (Fisher et al., J. Biol. Chem.265(4):2347-2351, 1990), BSP-KAE or OPN (Young et al. Genomics7:491-502, 1990; and Kiefer et al., Nucleic Acid Research 17:3306, 1989)cDNA into high expression, replication-deficient adenovirus (Ad5) usingEF-1 (BSP) and CMV (BSP-KAE, OPN, DMP1) promoters, respectively. TheRGD->KAE constructs were made using in situ mutagenesis and the entireinsert checked for fidelity. Adenoviruses were plaque-selected andpropagated on HEK 293 cells (ATCC #CRL1573). Cells were harvested whencytopathic effects were present and lysed by 5 freeze-thaw cycles.Cellular debris was removed and viral particles were purified by bandingtwice on CsCl. After dialysis in Tris/MgCl₂/Glycerol Buffer at 4° C.,viruses were aliquoted and frozen at −70° C. Evaluation of viral titerswas carried out by plaque formation of virus dilutions on HEK293 cells(Becker et al., Methods Cell Biol. 43(Pt A):161-189, 1994). Typically^(˜)24×10¹¹ pfu/mL were obtained from one viral preparation. Recombinantproteins were generated by infecting subconfluent normal human marrowstromal fibroblasts with 10,000 pfu/cell. Cells were maintained inalpha-MEM, 20% FBS and 100 IU/mL penicillin/100 μg/mL streptomycin in ahumidified atmosphere of 95% air and 5% CO2 at 37° C. Medium was changedto serum-free conditions after 48 hours. Subsequently, medium wascollected every 24-48 hours and frozen at −70° C. Aliquots were assayedby SDS-Gel electrophoresis and Western blot for BSP, DMP1, and OPNexpression. Expression was found to be at highest levels at ^(˜)168hours post infection. The proteins were purified by routine columnchromatography. Native BSP, BSP-KAE, DMP1, and OPN proteins werepurified by diluting medium from normal human marrow stromal fibroblastcells 1:1 with 40 nM phosphate buffer pH 7.4 and loading directly on a5.0×2.0 cm column packed with ToyoPearl TSK QAE resin. A linear saltgradient to 2.0 M NaCl was employed to purify the BSP, DMP1, and OPN to^(˜)95% purity as measured by SDS PAGE.

7. Biotinylated BSP

12 μg of recombinant human BSP was dissolved in 50 μL of PBS. 2 μL of afresh 1 mg/mL solution of NHS-LC-Biotin (Pierce Chemical Co., Chicago,Ill.) was added and the reaction incubated at room temperature for 45min. The unreacted biotin was removed by repeated washing with TBS-Tweenand centrifugation in a Microcon 30 (Amicon, Beverly, Mass.). A finalvolume of 50 μL was retained.

8. Alternate Complement Mediated Cell Lysis Assay

Murine erythroleukemia (MEL) cells (a gift of Dr. Marilyn Farquhar,Univ. CA San Diego.) grown in DMEM containing 10% fetal bovine serum and4 mM glutamine were rinsed three times with gelatin veronal buffer withMg₂ ⁺ and EGTA (GVB-MgEGTA, Sigma, St. Louis, Mo.). Cells wereresuspended in GVB-MgEGTA at a density of 5×10⁶ cells/mL and incubatedat 37° C. with different concentrations of normal human serum diluted inGVB-MgEGTA. After 2 h, cells were harvested for trypan blue exclusionassay by removing a 50 μL aliquot, incubating for 15 minutes in 0.4%trypan blue, and counting viable cells under an inverted microscope. Thethiazolium blue assay was carried out at identical serum dilutions byincubating a 50 μL aliquot of the cell suspension in an equal volume of1 mg/mL thiazolyl blue (MTT) for 45 min. Cell viability was determinedspectrophotometrically by absorbance at 560 nm. Cells in GVB-MgEGTAbuffer were pre-incubated with 10 μg of either rBSP, rDMP1, or rOPN in 1mL for 10 minutes at 37° C. Normal human serum collected for goodcomplement activity was then added at a dilution of 1:10 and the cellsreturned to 37° C. for 2 hours and cell viability was determined bytrypan blue-exclusion and MTT assays. For the assay of human cancer celllines, loosely adherent MCF-7 cells were selected for by sequentialgrowth in EMEM with 2 mM L-glutamine and Earle's balanced salt solutionadjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essentialamino acids and 1.0 mM sodium pyruvate and 10%; fetal bovine serum,while U-266 cells were cultured in RPMI 1640 medium containing 15% fetalbovine serum. Cells were collected by centrifugation and rinsed threetimes with GVB-MgEGTA buffer and subsequently treated exactly as for MELcells, substituting guinea pig serum (Sigma, St. Louis, Mo.) for humanserum.

EXAMPLES Example 1 Isolation of SIBLINGS Proteins and the RelativelyAcidic Form of BSP

A. Disruption of SIBLINGS/Factor H Complex

A variety of methods of disrupting protein-protein interactions are wellknown in the art. Such methods can involve treatment with harshchemicals and/or severe temperatures. Typically protein complexes suchas the SIBLINGS/Factor H complex can be disrupted through the use ofreducing agents, denaturants and/or increased temperatures. TheSIBLINGS/Factor H complex also can be disrupted using a combination ofreducing agents, denaturants, and increased temperatures. The particularmethod of disrupting the complex is not critical, as long as the complexis disrupted.

Reducing agents are agents that are capable of donating hydrogen atoms,and as such serve to disrupt disulfide bonds between amino acids. Theseagents particularly disrupt disulfide bonds that commonly link twoproteins or portions of the same protein together. Commonly usedreducing regents are β-mercaptoethanol, 1,4-dithiothieitol (DTT) andtrialkyl phosphines. Additionally, the ability of a reducing agent tofacilitate the separation of SIBLINGS proteins from Factor H will beincreased by increasing the temperature at which the solution containingthe reducing agent and SIBLINGS/Factor H complex is incubated.

Denaturants serve to relax the conformational structure of proteins, andthere are a variety of denaturants that can be employed to effectuatethe separation of SIBLINGS proteins from Factor H. Examples ofdenaturants that are particularly useful in conjunction with ionexchange columns are urea and formamide. However, denaturants such asguanidine hydrochloride or guanidine thiocyanate also may be useful forseparating Factor H from SIBLINGS proteins in non-ion exchange basedmethods. The ability of the denaturant to relax the protein will also beenhanced by increasing the temperature at which the sample containingthe SIBLINGS/Factor H complex is incubated.

SIBLINGS proteins, such as BSP can also be separated from Factor H byadding competitive binding agents to the sample containing theBSP/Factor H complex. Upon addition to the sample, a competitive bindingagent binds to Factor H and displaces BSP. Ideally, the agent used bindsto Factor H selectively and not to BSP. Examples of such competitivebinding agents are, recombinant fragments of BSP, sialic acids, andsynthetic BSP fragments.

Since a variety of methods can be employed to separate Factor H fromSIBLINGS proteins, the invention also provides a method of determiningwhen the proteins have become separated. As mentioned above, when FactorH is complexed to SIBLINGS proteins, the relatively acidic BSP, or tothe relatively non-acidic BSP, it masks the ionic charge normallyassociated with protein. Therefore, when the complex is placed on ananion exchange column, it readily flows through the column and thecomplex fails to bind. Hence, after treatment with one or moredenaturants and reducing agents, the sample can be tested for the itsability to bind to an anion exchange column. This assay can confirmwhether separation of Factor H from the complex has occurred.

It is also possible to simultaneously confirm that a SIBLINGS proteinhas been separated from Factor H and measure the SIBLINGS protein levelusing column chromatography. This can be done by first mixing a samplewith an anion exchange resin under conditions that disrupt the FactorH/SIBLINGS complex and then allowing the SIBLINGS protein to bind to theanion exchange resin. The un-complexed SIBLINGS protein will then bindto the resin, denaturants and other contaminants can be washed away, andthe SIBLINGS-resin complex can then be directly assayed using SIBLINGSprotein specific binding agents.

Additionally, the column conditions can be modified such that either therelatively acidic BSP or the relatively non-acidic BSP remains bound.Thus, BSP specific binding agents used to detect the BSP resin complexwill actually be detecting either the relatively non-acidic BSP or therelatively acidic BSP.

B. Separation of the Total Bound SIBLINGS protein from Factor H and theSeparation of the Relatively Non-Acidic BSP from the Relatively AcidicBSP

The separation of the total bound SIBLINGS protein from Factor H, andthe subsequent separation of the relatively acidic and the relativelynon-acidic BSP, can be accomplished using multi-step procedures thatinclude various chromatographic techniques (Robyt and White, BiochemicalTechniques Theory and Practice, Waveland Press, Inc, 1990). Suchmulti-step procedures will commonly operate to separate the proteins byexploiting their different physical characteristics. There are manydifferent methods that can be used to effectively separate SIBLINGSprotein from Factor H, as well as to separate the relatively acidic BSPfrom the relatively non-acidic SIBLINGS protein. Therefore, thefollowing discussion provides a broad description of variouschromatography techniques that can be used in either a single stepprocess or in multi-step process. Furthermore, it is possible that asingle variety of chromatography be repeatedly used to effectuate eitherthe isolation of the relatively acidic SIBLINGS protein, or the entirepopulation of SIBLINGS protein present in the sample.

1. Column Chromatography

One method of isolating the SIBLINGS protein and/or relatively acidicBSP is through adsorption chromatography. This method involves theexploitation of the differential affinity of the protein for the mediumin the column, and the affinity of the protein to the eluting solvent.An example of a suitable medium for use in the column is hydroxyapatite,which is a crystalline form of calcium phosphate. This medium tends toadsorb acidic proteins (such as BSP), which can subsequently be elutedwith phosphate ions, which have a high affinity for the calcium ionspresent in the hydroxyapatite.

Ion exchange chromatography can also be used to isolate SIBLINGSprotein, and/or the relatively acidic BSP. This approach is a variationof adsorption chromatography, in which the solid adsorbent has chargedgroups chemically linked to an inert solid. The ionic charge of theSIBLINGS protein then causes the SIBLINGS protein to become linked tothe chemically charged groups on the solid support, and the SIBLINGSprotein is subsequently released by passing a solution containing an iongradient over the solid adsorbent. Examples of solid supports that canbe used in this technique include DEAE-cellulose, DEAE-Sephadex,DEAE-Bio-Gel, DEAE cellulose, DEAE sepharose, DEAE sephacryl, DEAEtrisacryl, Q sepharose, ecteola cellulose, QAE cellulose, express ionexchanger Q, PEI cellulose, and other polystyrene-based anionexchangers.

Another type of adsorption chromatography which can be used to isolateSIBLINGS protein and/or the relatively acidic form of BSP is affinitychromatography. This method involves covalently linking to an inertsolid support a ligand which has binding affinity for the protein ofinterest, which in this case is either SIBLINGS protein or therelatively acidic BSP. Commonly, the ligand is a specific binding agent,which selectively binds the protein of interest as it contacts the solidsupport. Alternatively, the SIBLINGS protein and/or the relativelyacidic BSP can be separated based upon hydrophobicity. For example,alkyl chains may be linked to the inert support, to supply sites forhydrophobic bonding interactions. The eluting solvent then contains ahydrophobic gradient.

High performance liquid chromatography provides yet another method thatcan be utilized to isolate SIBLINGS protein and/or the relatively acidicBSP. All of the major classes of chromatographic separations arepossible using this method, for example, adsorption, liquid-liquidpartition, ion exchange, exclusion, and affinity chromatography can beused in conjunction with HPLC. Additionally, the HPLC method allows forreversed phase and ion pair partition. Reversed phase partition allowsfor quick elution using a non-polar stationary phase and a polar mobilephase, while ion pair partition involves pairing the charged polarsubstance with its counter ion to create a less polar species that thenflows through the column.

2. Electrophoresis

Electrophoresis is a well-established technique for the separation andanalysis of mixtures by differential migration and separation ofmolecules in an electric field, based on differences in mobility througha support. Many different forms of electrophoresis have been developedto permit the separation of different classes of compounds. These formsinclude paper and cellulose acetate electrophoresis, thin-layerelectrophoresis, gel electrophoresis, immunoelectrophoresis andisoelectric focusing. Paper electrophoresis operates best for theseparation of relatively low molecular weight protein molecules, and gelelectrophoresis is a much better candidate for use in isolating therelatively acidic BSP. There are a variety of different matricesavailable for forming gels, but matrices with a relatively smaller poresize are most suitable for the separation of two similar molecules.Furthermore, proteins may be separated based upon size by conducting theelectrophoresis under disassociating conditions, for example using SDS.The SDS relaxes the protein and masks the ionic charge of the protein,which leaves protein size as the only distinguishing characteristic.Hence in the case of the relatively acidic BSP, SDS would not be usedfor separating the two populations of BSP (the relatively acidic versusthe relatively non-acidic), because the separation will depend on theionic charge that would be masked by SDS.

Separation can also be achieved using immunoelectrophoresis, in whichproteins are separated on a gel based upon their charge-to-mass ratioand their antigenicity. This is done by first separating the proteins ona gel, and then adding an antibody specific for the protein of interestto a well created in the gel, and allowing the antibody to diffusethrough the gel. Precipitate then forms in regions in which the antibodyreacts with the protein.

The relatively acidic form of BSP can also be isolated through the useof isoelectric focusing. In this type of electrophoresis the protein isplaced on a substrate having a pH gradient, and it will move under theinfluence of an electrical field until it reaches an equilibrium pointwith the pH gradient.

Finally, the characterization of the relatively acidic BSP populationindicates that other SIBLINGS proteins may exist in multiple forms.Moreover, SIBLINGS proteins may also have relatively acidic populationand these populations can be detected using the above describedtechniques.

Example 2 Identifying SIBLINGS Proteins with Specific Binding Agents

A SIBLINGS protein can be identified using specific binding agents.These specific binding agents may bind for example, BSP generally, orthey may selectively bind to the relatively acidic BSP, the relativelynon-acidic BSP, or the exposed portion of SIBLINGS protein in theSIBLINGS/Factor H complex. The latter specific binding agent will beparticularly useful for more accurately determining the total amount ofSIBLINGS protein in the biological specimen without the need to separatethe SIBLINGS protein from the Factor H.

A. Antibodies

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays such as ELISAs, Western blotting and RIAs,to determine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological specimen.

1. Production of an Antibody to SIBLINGS Proteins

Monoclonal or polyclonal antibodies may be produced to SIBLINGS proteinsor portions thereof. Optimally, antibodies raised against a particularSIBLINGS protein, such as BSP will specifically detect BSP. That is,antibodies raised against BSP would recognize and bind BSP and would notsubstantially recognize or bind to other proteins found in human cells.The determination that an antibody specifically detects a SIBLINGSprotein, such as BSP is made by any one of a number of standardimmunoassay methods; for instance, the Western blotting technique(Sambrook et al., In Mol. Clon.: A Lab. Man., Cold Spring Harbor, N.Y.,1989). To determine that a given antibody preparation (such as oneproduced in a mouse against the human BSP) specifically detects BSP byWestern blotting, serum is extracted from a subject (for example, toobtain lymphocytes) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are then transferred to amembrane (for example, nitrocellulose or nylon) by Western blotting, andthe antibody preparation is incubated with the membrane.

After washing the membrane to remove non-specifically bound antibodies,the presence of specifically bound antibodies is detected by the use ofan anti-mouse antibody conjugated to an enzyme such as alkalinephosphatase; application of the substrate 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium results in the production of a denseblue compound by immuno-localized alkaline phosphatase. Antibodies thatspecifically detect SIBLINGS protein will, by this technique, be shownto bind substantially only the SIBLINGS protein band (which will belocalized at a given position on the gel determined by its molecularweight). Non-specific binding of the antibody to other proteins mayoccur and may be detectable as a weak signal on the Western blot. Thenon-specific nature of this binding will be recognized by one skilled inthe art by the weak signal obtained on the Western blot relative to thestrong primary signal arising from the specific antibody-SIBLINGSprotein binding.

Antibodies that specifically bind to SIBLINGS protein belong to a classof molecules that are referred to herein as specific binding agents.Specific binding agents that are capable of specifically binding toSIBLINGS protein may include polyclonal antibodies, monoclonalantibodies (including humanized monoclonal antibodies) and fragments ofmonoclonal antibodies such as Fab, F(abγ)2 and Fv fragments, as well asany other agent capable of specifically binding to SIBLINGS proteins.

Depending on the desired specificity of the antibody, different forms ofa SIBLINGS protein can be used as immunogens for injection into subjectsfor the creation of antibodies. To create antibodies which recognize theexposed portion of a SIBLINGS protein when it is bound to Factor H, theentire SIBLINGS/Factor H complex can be used as an immunogen. Similarly,if the creation of an antibody specific for the relatively acidic BSP isdesired, the relatively acidic BSP can be isolated and used to inoculatea mouse. Additionally, as mentioned below, fragments of the relativelyacidic BSP or fragments of the exposed portion of BSP can be used asimmunogens for the creation of specific binding agents.

Regardless of the particular form of SIBLINGS protein used to create theimmunogen the concentration of protein in the final preparation isadjusted, for example, by concentration on an Amicon filter device, tothe level of a few micrograms per milliliter. Alternatively, aspreviously mentioned, peptide fragments of the SIBLINGS proteins may beutilized as immunogens. Such fragments may be chemically synthesizedusing standard methods, or may be obtained by cleavage of the wholeSIBLINGS protein followed by purification of the desired peptidefragments. Peptides as short as 3 or 4 amino acids in length areimmunogenic when presented to the immune system in the context of aMajor Histocompatibility Complex (MHC) molecule, such as MHC class I orMHC class II. Accordingly, peptides comprising at least 3 and preferablyat least 4, 5, 6 or more consecutive amino acids of the disclosedSIBLINGS protein amino acid sequences may be employed as immuogens toraise antibodies.

Because naturally occurring epitopes on proteins frequently includeamino acid residues that are not adjacently arranged in the peptide whenthe peptide sequence is viewed as a linear molecule, it may beadvantageous to utilize longer peptide fragments from the SIBLINGSprotein amino acid sequence in order to raise antibodies. Thus, forexample, peptides that comprise at least 10, 15, 20, 25 or 30consecutive amino acid residues of the BSP amino acid sequence may beemployed. Monoclonal or polyclonal antibodies to the intact BSP orpeptide fragments of this protein may be prepared as described below.

2. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of BSP identified and isolated asdescribed can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein, Nature 256:495, 1975, orderivative methods thereof. Briefly, a mouse is repetitively inoculatedwith a few micrograms of the selected protein over a period of a fewweeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media such as aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate, where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, Enzymol. 70:419, 1980, and derivative methods thereof. Selectedpositive clones can be expanded and their monoclonal antibody productharvested for use. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Antibodies, A Lab. Man.,Cold Spring Harbor, N.Y., 1988. In addition, protocols for producinghumanized forms of monoclonal antibodies (for therapeutic applications)and fragments of monoclonal antibodies are known in the art.

3. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Forexample, small molecules tend to be less immunogenic than others and mayrequire the use of carriers and adjuvant. Also, host animals vary inresponse to site of inoculations and dose, with both inadequate orexcessive doses of antigen resulting in low titer antisera. Small doses(ng level) of antigen administered at multiple intradermal sites appearto be most reliable. An effective immunization protocol for rabbits canbe found in Vaitukaitis et al., J. Clin. Endocrinol. Metab. 33:988-991,1971.

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al., In Handbook of Experim. Immunol., Wier, D.ed., ch. 19. Blackwell, 1973. Plateau concentration of antibody isusually in the range of 0.1 to 0.2 mg/mL of serum (about 12 M). Affinityof the antisera for the antigen is determined by preparing competitivebinding curves, as described, for example, by Fisher, 1980.

4. Antibodies Raised by Injection of SIBLINGS Protein cDNA

Antibodies may also be raised against SIBLINGS proteins by subcutaneousinjection of a DNA vector that expresses the SIBLINGS protein inlaboratory animals, such as mice. Delivery of the recombinant vectorinto the animals may be achieved using a hand-held form of the Biolisticsystem (Sanford et al., Particulate Sci. Technol. 5:27-37, 1987) asdescribed by Tang et al., Nature (London) 356:152-154, 1992. Expressionvectors suitable for this purpose may include those that express a cDNAencoding a SIBLINGS protein under the transcriptional control of eitherthe human β-actin promoter or the cytomegalovirus (CMV) promoter.Methods of administering naked DNA to animals in a manner to causeexpression of that DNA in the body of the animal are well known and aredescribed, for example, in U.S. Pat. No. 5,620,896 (DNA vaccines againstrotavirus infection), U.S. Pat. No. 5,643,578 (Immunization byinoculation of DNA transcription unit) and U.S. Pat. No. 5,593,972(Genetic immunization), and references cited therein.

5. Antibody Fragments

Antibody fragments may be used in place of whole antibodies and may bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as antibody fragments, are well known and include thosedescribed in Better and Horowitz, Advances in Gene Technol.: The Mol.Biol. Of Immune Disease & the Immune Response (ICSU Short Reports),1989; Glockshuber et al., Biochem. 29:1362-1367 (A Comparison ofStrategies to Stabilize Immunoglobulin F, Fragments), 1990; and U.S.Pat. No. 5,648,237 (Expression of Functional Antibody Fragments), U.S.Pat. No. 4,946,778 (Single Polypeptide Chain Binding Molecules), andU.S. Pat. No. 5,455,030 (Immunotherapy Using Single Chain PolypeptideBinding Molecules), and references cited therein.

6. Humanized Antibodies

Humanized monoclonal antibodies are preferred in clinical applications.Methods of making humanized monoclonal antibodies are well known, andinclude those described in U.S. Pat. No. 5,585,089 (HumanizedImmunoglobulins), U.S. Pat. No. 5,565,332 (Production of ChimericAntibodies—A Combinatorial Approach), U.S. Pat. No. 5,225,539(Recombinant Altered Antibodies And Methods Of Making AlteredAntibodies), U.S. Pat. No. 5,693,761 (Polynucleotides Encoding ImprovedHumanized Immunoglobulins), U.S. Pat. No. 5,693,762 (HumanizedImmunoglobulins), U.S. Pat. No. 5,585,089 (Humanized Immunoglobulins),and U.S. Pat. No. 5,530,101 (Humanized Immunoglobulins), and referencescited therein.

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays to determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample.

Example 3 Characterizing SIBLINGS Protein Populations in BiologicalSpecimens

In addition to the assays provided above, the present invention allowsfor further characterization of SIBLINGS protein populations inbiological specimens, and the development of screening assays that candetect tumors, as well as abnormal bone turnover. It is additionallycontemplated that tumors such as sarcomas and carcinomas, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, bladder carcinoma, CNS tumors (such as a glioma,astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma and retinoblastoma) may prove to express BSP andbe detectable through the use of the disclosed assays.

A. Development of Screening Assays

The invention allows for the development of screening assays that candetect other disease states which are associated with elevated SIBLINGSprotein levels, and/or shifts in the population of BSP from therelatively non-acidic BSP to the relatively acidic form of BSP. This canbe done by separating the SIBLINGS protein from Factor H and correlatingthe relative concentration of SIBLINGS protein in the test sample to therelative concentration in a control. The control in many cases is a likesample from a subject that does not have the particular malady inquestion. For example, a screening assay can be developed to detectabnormal bone turnover in subjects having osteoporosis orosteoarthritis, by taking a sample from the subject that is know to havethe particular malady, and characterizing the SIBLINGS proteinconcentration using the methods provided above. This same procedure canbe then be used to characterize a sample from a normal subject andsubsequently a correlation can be drawn between the results from the twosubjects. If present the correlation can then be used to detect themalady in other subjects.

B. Controls for Assays in which the Relatively Acidic BSP is Used as aMarker

The above data shows subjects suffering with breast, prostate, thyroid,multiple myeloma and lung tumors have readily distinguishablepopulations of BSP. These differences have permitted the development ofan assay that can be used to detect the presence or recurrence of suchtumors (such as recurrence following surgical excision of a tumor).Generally, such assays include a control that allows a comparison to bemade between a normal individual and the test subject. However, in somecases this control can be recombinantly expressed BSP, or the testsample can be classified as positive for a tumor based upon the ratio ofthe relatively non-acidic BSP to the relatively acidic BSP. The aboveseparation methods describe different mechanisms for separating proteinsbased upon ionic charge and these methods can be used to provideinformation on the relative concentration of the relatively non-acidicform of BSP, as well as the relatively acidic form of BSP. Therefore,used in this way the assay does not require the use of an externalcontrol; rather the ratio itself can be used to indicate the presence ofa tumor.

Example 4 Use of Members of the SIBLINGS Family of Proteins to ConferProtection Against Complement

The above-described methods and results show that when members of theSIBLINGS family of proteins are associated with cells they can serve toprotect the cells from complement mediated lysis. In the case of tumorcells it is likely that the expression of SIBLINGS proteins serves toprotect the tumor from complement lysis. This protection may beespecially important during the process of metastasis when tumor cellsare invading other tissue such as bone.

The complement protective activity of SIBLINGS proteins can also beexploited to protect cells and/or implants from complement mediatedimmune responses. In the case of implants SIBLINGS proteins can be usedto coat implants such as described in U.S. Pat. No. 6,024,918 toHendriks, et al. or they can be delivered from a reservoir that isplaced in the body. This protection can be imparted to a cell by eithercoating the cell with the SIBLINGS protein, or genetically engineering acell to express the SIBLINGS protein. Used in this way a cell that hasbeen engineered to express a transgene is designed to also express oneor more of the members of the SIBLINGS family of proteins. Theco-expression of the SIBLINGS protein then facilitates the binding ofFactor H to the transgenic cell. This binding results in the cellbecoming protected from complement mediated lysis. Expressing theSIBLINGS protein with a membrane localization peptide could furtherenhance the protection conferred to the transgenic cell.

The complement protective activity of SIBLINGS proteins also could beused to provide protection against non-self cells that are introducedinto a subject's body, for example by implantation of a graft. Used inthis way a transgene encoding the SIBLING protein could be delivered tothe graft via various vectors such as described below.

A. Expression Cassette Transfer

A preferred method of supplying exogenous SIBLINGS proteins is bytransferring a vector comprising an expression cassette to cellsassociated with the region of interest such that the cells express theSIBLINGS protein.

1. Expression Cassettes

Expression cassettes employed in the present inventive methods are ofany type appropriate for cells containing the cassette to express theprotein of interest. Thus, for example, an expression cassette comprisesa polynucleotide encoding BSP operably linked to a promoter.

Any promoter and/or enhancer sequence appropriate for controllingexpression of polynucleotides from the vector can be used inconstructing an expression cassette. While such promoters/enhancerelements are well known in the art, examples of suitable promotersinclude prokaryotic promoters or viral promoters, (e.g., ITRs, or LTRs;immediate early viral promoters, such as herpesvirus IE promoters, orcytomegalovirus (CMV) IE promoters; or other viral promoters, such asRous Sarcoma Virus (RSV) promoters, or Murine Leukemia Virus (MLV)promoters). Other suitable promoters are eukaryotic promoters, such asconstitutively active promoters (e.g., the β-actin promoter), signalspecific promoters (e.g., inducible promoters, such as a promoterresponsive to TNF), or tissue-specific promoters, (e.g., those active inepidermal tissue, dermal tissue, tissue of the digestive organs such ascells of the esophagus, stomach, intestines, or colon), smooth muscles,such as vascular smooth muscles, cardiac muscles, skeletal muscles, lungtissue, hepatocytes, lymphocytes, endothelial cells, sclerocytes, kidneycells, glandular cells (e.g., those in the thymus, ovaries, testicles,pancreas, adrenals, pituitary, etc.), tumor cells, cells in connectivetissue, cells in the central nervous system (e.g., neurons, neuralgia,etc.), cells in the peripheral nervous system, or other cells ofinterest.

Which promoter is used in a given expression cassette will depend, inpart, on the choice of vector. Thus, for example, an expression cassettecan comprise a native retroviral LTR promoter operably linked to acoding polynucleotide when the vector is a retroviral vector.

In addition to a promoter, an expression cassette will also contain apolynucleotide encoding the protein of interest. Preferably, thepolynucleotide is a synthetic DNA, cDNA or genomic DNA fragment encodingthe SIBLINGS protein or fragment thereof that imparts protection againstcomplement mediated lysis to the cell that expresses it. Expressioncassettes can also include other polynucleotides, such as apolyadenylation sequence. Also, expression cassettes can encode morethan one SIBLINGS protein.

2. Vectors

An expression cassette for use in the present inventive methods such asthose described supra is contained within a vector. Of course, inaddition to the SIBLINGS protein encoding sequence, the vector caninclude other expression cassettes, such as, for example, cassettes forexpressing a selectable marker (e.g., β-gal or a marker conferringresistance to a toxin), a pharmacologically active protein, atranscription factor, or other biologically active substances.

Any vector appropriate for transferring an exogenous expression cassetteto a cell is included within the scope of the present inventive methods.Preferably, the vector is a viral vector. Examples of viral vectorsemployed in accordance with the present inventive method include, butare not limited to, retroviral vectors, adenoviral vectors,adeno-associated viral vectors, herpesviral vectors, SV40 viral vectors,polyoma virus vectors, pappiloma virus vectors, picnoravirus vectors,vaccinia virus vectors, or other suitable vectors.

In addition to viral vectors, any non-viral vector capable of expressionupon infection of target cells can be used in the present inventivemethods. Preferably, a non-viral vector is a plasmid.

The skilled artisan will be able to incorporate an expression cassetteinto the nucleic acid sequence of the vector. Methods for incorporatingexpression cassettes into viral vectors are well known in the art (seee.g., Sambrook, et al. Molecular Cloning: a Laboratory Manual, 2dedition, Cold Spring Harbor Press, 1989) and include direct cloning,site specific recombination using recombinases, such as the flprecombinase or the cre-lox recombinase system (reviewed in Kilby et al.,Trends in Genetics, 9:413-21, 1993), homologous recombination, and othersuitable methods of constructing a recombinant vector.

3. Target Cells

The present inventive methods also comprise transferring an expressioncassette to cells associated with the region of interest (i.e., agraft). Any suitable cells associated with the region of interest whichare capable of supporting expression of the coding polynucleotide of thecassette are encompassed within the present inventive methods. Suchcells can be cells in situ, such as cells of the graft tissue.

Following transfer of the vector comprising the expression cassette tothe cells in vitro, the cells associated with the region are transferredto the region of interest. Transfer of cells in vitro to a region ofinterest is accomplished by known means. For example, cells can betransferred to internal tissue of a patient by methods described herein.Further methods of transferring cells containing an expression cassetteare described in International Patent Application No. WO 96/00006,(Billiar et al.).

4. Vector Transfer

The present inventive methods comprise transferring a vector comprisingan expression cassette, such as those described herein, to cellsassociated with the region of interest (e.g., graft). Any method oftransferring the expression cassette to the cells is appropriate so longas a product of the expression cassette is produced in the cells.

a. Non-Viral Vectors

Any means of introducing non-viral vectors into target cells, such as bydirect DNA injection, electroporation, calcium-phosphate mediated celltransfection, lipofectamine, DAEA-dextran-mediated cell transfection,polybrene-mediated delivery, host cell fusion, microinjection, andpolylysine-mediated cell transfection is appropriate within the presentinventive context. Such methods are well known in the art, and some aredescribed in International Patent Application No. WO 96/00006, (Billiaret al.). A preferred method of transferring the expression cassettewithin a nonviral vector, such as a plasmid, is via liposome-mediatedtransfection of the target cells, such as endothelial cells, in vitro orin situ. If the vector is transfected in vitro, the cells associatedwith the region of interest are subsequently transferred to the patient.

b. Viral Vectors

Transfer of an expression cassette to cells associated with the regionof interest where the cassette is within a viral vector is accomplishedby infecting the cells with the virus. Where the virus is a retrovirus,the virus can be first transfected into an appropriate packaging cellline for generation of infectious virus. Cells associated with theregion of interest can be infected in vitro or in situ. Moreover, themode of transfer of the expression cassette does not depend on theidentity of the coding polynucleotide.

c. In Vitro Delivery

Infectious viruses are used to infect cultured cells in vitro. Theprecise method for infecting target cells in vitro will depend upon theviral vector used; however, such methods are well known in the art. Somesuitable methods are described in International Patent Application No.WO 96/00006, (Billiar et al.).

5. In Situ Delivery

Some applications of the present inventive methods involve transfer ofexpression cassettes in situ. In situ transfer can be either in vivo(i.e., to a wound or to a graft following implantation), or ex vivo(i.e., to a graft prior to implantation). Any method of delivering thevector comprising the expression cassette to cells associated with theregion of interest in situ is within the present methods. Such methodsalso apply to transfer of exogenous cells to the region of interest.Methods for in situ delivery of vectors preferably involve physicallysegregating the region of interest from the remainder of the patient'stissue in order to properly target the vector within the region. Uponsegregation, the vector is applied to the region of interest in a mannerappropriate to transfer the expression cassette into the cellsassociated with the region of interest.

Tissue ex vivo, such as, for example, a graft, is completely isolatedfrom the patient. For ex vivo vector transfer, the vector can be appliedto the tissue in any suitable manner, e.g., in a carrier appropriate fortransferring the vector. For example, the tissue can be incubated in acarrier containing the vector particles by any suitable incubationmethod. Other tissue can be perfused with the solution containing thevector.

The vectors or exogenous cells preferably remain in contact with thewound or tissue for a period of time sufficient to promote the transfer.For example, a carrier comprising a vector will remain in contact withthe wound or ex vivo tissue from about one minute to about 2 hours, morepreferably between 10 minutes and an hour, and most preferably fromabout 20 minutes to 40 minutes. In many applications, the carrier willoptimally remain in contact with the wound or tissue for about 30minutes.

6. Internal Delivery

For in situ delivery of a vector internally, the region of interestdesirably is further segregated from the remainder of the subject'stissue. Any of a variety of known surgical procedures for physicallysegregating the region of interest is appropriate. Various endovascularsurgical techniques appropriate for segregating a region of interest areavailable, depending upon the location of the target.

Endovascular surgical procedures include, but are not limited to,balloon angioplasty, intravascular stents, laser-assisted balloonangioplasty, double balloon catheterization, mechanical endarterectomyand vascular endoscopy. For a review of endovascular alternatives, seegenerally Ahn, “Endovascular Surgery,” in Vascular Surgery, AComprehensive Review, Ed. W. S. Moore, W. B. Saunders & Co.,Philadelphia, 1993.

Several catheter designs can be utilized for local delivery of a vector.One catheter design consists of two independently inflated balloons, oneproximal and one distal to the vascular delivery site. Inflation ofthese balloons provides an evacuated isolated arterial segment intowhich vectors for expression cassette delivery can be infused. Thissystem is, however, limited by absence of distal arterial perfusion. Asecond catheter design developed by Wolinsky allows the infusion of theSIBLINGS protein-encoding vector through 25-100 μM pores under pressuresup to 5 atm. This perfusion pressure increases the depth of penetrationby the vectors and additionally increases expression cassette transferefficiency. Yet another catheter design utilizes an expandable stentwhich traps the balloon against the arterial wall and allows intramuraldelivery of the expression cassette through spaces in the stentmaterial. Additionally, these stents can be modified with burrs thatcreate holes deeper in the vessel wall and allow flow of the expressioncassette delivery agents to these sites to allow more uniform deliveryof the expression cassette throughout the vessel wall.

Another delivery mechanism is to coat the catheter with a hydrophilicpolyacrylic acid polymer that acts as a drug-absorbing sponge. Bydisrupting the vessel during the angioplasty procedure, this hydrogel isdeposited within the vessel wall and will allow sustained delivery ofthe vector at the arterial wound site. Additionally, the iontophoreticballoon catheter is a catheter design that uses low electrical currentto change the cell membrane polarity and allow the diffusion of chargedDNA particles into the cell. This is a potential delivery mechanism forplasmid DNA expression cassette constructs. Also, biodegradable stentsformed from agents such an ethylenevinyl acetic copolymer areappropriate for localized delivery to vascular tissue. Alternatively, anintravascular stent can be utilized wherein the endovascular scaffold ofthe stent is bathed in a ointment, cream, lotion, colloidal dispersionsuch as a gel or magma or any other acceptable carrier which comprisesthe SIBLINGS protein encoding vector for delivery to the targetedportion of a vessel segment. This solution is applicable to either an insitu or ex vivo based vessel delivery.

Example 5 Additional Examples of Methods for Detecting and/orQuantifying SIBLINGS Proteins

The following assays describe the detection of BSP, however, theseassays, with appropriate modifications (i.e., antibody substitutions),can also be used to detect other SIBLINGS proteins.

A. Chemiluminescent Sandwich Assay for BSP in Human Serum

Pierce Reacti-Bind White Opaque 96-Well EIA plates (product # 15042) arecoated with 100 μL of 1 μg/mL Goat anti Mouse IgG (H & L), human serumadsorbed (Kirkegaard & Perry, Gaithersburg, Md.) in PBS. The plates arethen covered and incubated overnight at 4° C. The next morning the wellsare emptied and 250 μL of 1% bovine serum albumin is added for 15 min.The albumin is then removed and the plates are washed 3 times with^(˜)300 μL PBS-0.05% Tween 20 (PBS-T) for 5 minutes each at roomtemperature. 100 μL 1:1000 LFMb-2 (monoclonal to human BSP) in PBS-T isadded to each well and the plate is incubated again for 1 hour at roomtemp and then washed three times (PBS-Tween).

The samples are prepared from 25 μL human. 50 μL fresh formamide and 1μL of 0.1 M Dithiothreitol is added to the sample and the sample is thenheated at 100° C. for 5 minutes and diluted to 1 mL with PBS-Tcontaining 0.03% H₂O₂. This addition dilutes the fomamide to levelssufficiently low enough to promote normal antibody binding and the H₂O₂inactivates the remaining DTT.

The BSP standard (5 ng to 0.15 ng) and a negative control (without BSP)are prepared in PBS-T and added to individual wells. 100 μL dilutedsample is also added to the individual wells. The samples and thestandards are then incubated 1 hour at room temperature and then washed3 times with PBS-Tween. 100 μL of 1:1000 LF-100 polyclonal (rabbitanti-human BSP) antibody in PBS-T plus 1% normal mouse serum is thenadded to each well and the plate is incubated for 1 hour at roomtemperature and then washed 3 times. 100 μL of 1:50,000 dilutedperoxidase-conjugated goat anti rabbit IgG (H&L), multi-serum adsorbed(Kirkegaard & Perry) is then added to each well and the plate isincubated for 1 hour at room temperature, washed 3 times (PBS-Tween).

The results are then visualized by adding 100 μL of Pierce SupersignalELISA Pico Chemiluminescent Substrate (product # 37070) and incubating10 minutes at room temperature. Luminescence can then be detected in aluminometer.

B. Western Blot and Quantitative Immunoanalysis

5 μL of serum is added to 95 μL of sample buffer (0.0625 M Tris, pH 6.8,2.3% SDS, 5% 2-mercaptoethanol or 1 mM dithiothreitol, 8 M urea or 50%formamide, bromophenol blue tracking dye), the sample is then heated for5 minutes at 100° C. 10 μL of the sample is then added to each well of a4-20% or 4-12% gradient acrylamide gel (Novex Corp) and the gel iselectrophoresed with 100-200 V constant voltage until the dye frontreaches the bottom of the gel. Molecular weight standards (prestained)and BSP standards are also electrophoresed.

The protein is then transferred to nitrocellulose or other western blotmembranes under standard conditions. If nitrocellulose is used transfercan be achieved in 60 minutes at 100 volts with cooling in the NovexXCell II Blot Module in Tris/Glycine buffer containing 20% methanol.

Protein is then detected by blocking the membrane with 3% BSA-NGS in PBSfor 15 minutes and then adding 1% normal goat serum for 45 minutes. Anantibody, for instance the polyclonal antibodye LF-100, or monoclonalLF-Mb-11 is then diluted at 1:2000 and added to the membrane for 1 hourat room temperature. The membrane is then washed 3 times with PBS-Tween20 (0.05%). 3% BSA-1% NGS is added and then HRP-conjugated secondantibody (goat anti-rabbit or goat anti-mouse IgG respectively) at isadded to a final concentration of 1:50,000. The membrane is thenincubated for 1 hour at room temperature and washed 3 times. Finally,Pierce SuperSIgnal West Pico Chemiluminescent reagent is added for 5minutes and the membrane is placed in clear plastic bag and exposed to achilled digital camera (Fuji LAS-100).

In view of the many possible embodiments to which the principles of theinvention may be applied, it should be recognized that the illustratedembodiments are only particular examples of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1-14. (canceled)
 15. A composition comprising a specific binding agentwhich specifically binds at least one SIBLINGS protein when the SIBLINGSprotein is complexed with Factor H.
 16. The composition of claim 15wherein the specific binding agent is a monoclonal antibody.
 17. Thecomposition of claim 16 wherein the monoclonal antibody specificallyrecognizes the binds an exposed portion of the SIBLINGS protein when theSIBLINGS protein is complexed with a Factor H protein.
 18. A hybridomawhich expresses the monoclonal antibody of claim
 17. 19. (canceled) 20.A method of making the monoclonal antibody of claim 16, comprisingpresenting an antigen which is unique to a SIBLINGS/Factor H complex toa subject.
 21. An antibody made by the method of claim
 20. 22-23.(canceled)
 24. A purified relatively acidic BSP produced by an organismof claim
 23. 25. A method of conferring protection from a complementmediated immune response to a subject, the method comprising providingwithin a body a supply of a protective amount of a protein selected fromthe group consisting of a SIBLINGS protein and a biologically activefragment of a SIBLINGS protein, sufficient to protect the subject from acomplement immune response by association with the protein.
 26. A methodof conferring protection from complement mediated lysis to a cell, themethod comprising: providing a vector encoding at least one biologicallyactive SIBLINGS protein; transforming the cell with the vector, whereinthe resulting transformed cell expresses the biologically activeSIBLINGS protein, and such expression confers resistance to complementmediated lysis to the cell.
 27. The method of claim 25, wherein theSIBLINGS protein is BSP.
 28. The method of claim 25, wherein theSIBLINGS protein is OPN.
 29. The method of claim 25, wherein theSIBLINGS protein is DMP1.
 30. The method of claim 25, wherein theSIBLINGS protein is DSPP.
 31. The method of claim 26 wherein the cell isa cell found in a graft.
 32. The method of claim 26 wherein the cell isbone marrow cell. 33-34. (canceled) 36-45. (canceled)